Pantropic neurotrophic factors

ABSTRACT

Pantropic neurotrophic factors which have multiple neurotrophic specificities are provided. The pantropic neurotrophic factors of the present invention are useful in the treatment of neuronal disorders. Nucleic acids and expression vectors encoding the pantropic neurotrophins are also provided.

This is a continuation of application Ser. No. 08/253,937 filed on Jun.3, 1994, abandoned, which application is incorporated herein byreference and to which application priority is claimed under 35 USC§120.

FIELD OF THE INVENTION

This application relates to proteins which are involved in the growth,regulation or maintenance of nervous tissue, particularly neurons. Inparticular, it relates to pantropic neurotrophic factors which havemultiple neurotrophic specificities (MNTS variants).

BACKGROUND OF THE INVENTION

The survival and maintenance of differentiated function of vertebrateneurons is influenced by the availability of specific proteins referredto as neurotrophins. Developing neurons depend for survival on thesupply of these factors from their target fields and the limitedproduction of neurotrophins results in death of superfluous neurons (forreviews, see (1); (2)). The various neurotrophins differ functionally intheir ability to support survival of distinct neuronal populations inthe central and the peripheral nerve system (3), (4); (5), (80).

The neurotrophin family is a highly homologous family which includes NT3(6), (7); (5); (8); (9); (10), nerve growth factor (NGF) (11); (12),brain-derived neurotrophic factor (BDNF) (13); (14)) and neurotrophin4/5 (NT4/5) ((15), (16), (17).

Studies suggest that neurotrophins transduce intracellular signalling atleast in part through the ligand-dependent activation of a class oftyrosine kinase-containing receptors of M_(r) =140-145,000 known as thetrks (18); (19) (21); (20) (22); (23); (24); (25); (26). Thus, thesignal transduction pathway of neurotrophins is initiated by thishigh-affinity binding to and activation of specific tyrosine kinasereceptors and subsequent receptor autophosphorylation (19); (27).Although there is some degree of cross-receptor interaction between theneurotrophins and the different trks, the predominant specificityappears to be NGF/trkA, BDNF/trkB, and NT3/trkC while NT4/5 appears tointeract primarily with trkB as efficiently as BDNF (27); (19) (21);(25); (22); (28); (18); (28a). While trkC responds exclusively to NT3(25); (26), trkA and trkB can respond in vitro under certaincircumstances to multiple neurotrophins (6); (23). However, the neuronalenvironment does restrict trkA and trkB in their ability to respond tonon-preferred neurotrophic ligands (29). In addition to the trk familyof receptors, the neurotrophins can also bind to a different class ofreceptor termed the p75 low affinity NGF receptor (p75; (30); (31))which has an unknown mechanism of transmembrane signalling but isstructurally related to a receptor gene family which includes the tumornecrosis factor receptor (TNFR), CD40, OX40, and CD27 (32); (33); (34),(35); (36); (37)). The role of the gp75 in the formation ofhigh-affinity binding sites and in the signal transduction pathway ofneurotrophins is as yet unclear (for reviews see (38); (39)).

An examination of the primary amino acid sequence of the neurotrophinsreveals seven regions of 7-10 residues each which account for 85% of thesequence divergence among the family members.

Nerve growth factor (NGF) is a 120 amino acid polypeptide homodimericprotein that has prominent effects on developing sensory and sympatheticneurons of the peripheral nervous system. NGF acts via specific cellsurface receptors on responsive neurons to support neuronal survival,promote neurite outgrowth, and enhance neurochemical differentiation.NGF actions are accompanied by alterations in neuronal membranes (40),(41), in the state of phosphorylation of neuronal proteins (42), (43),and in the abundance of certain mRNAs and proteins likely to play a rolein neuronal differentiation and function (see, for example (44)).

Forebrain cholinergic neurons also respond to NGF and may require NGFfor trophic support. (45). Indeed, the distribution and ontogenesis ofNGF and its receptor in the central nervous system (CNS) suggest thatNGF acts as target-derived neurotrophic factor for basal forebraincholinergic neurons (46), (81).

Little is known about the NGF amino acid residues necessary for theinteraction with the trkA-tyrosine kinase receptor. Significant lossesof biological activity and receptor binding were observed with purifiedhomodimers of human and mouse NGF, representing homogenous truncatedforms modified at the amino and carboxy termini. (47); (48); (49). The109 amino acid species (10-118)hNGF, resulting from the loss of thefirst 9 residues of the N-terminus and the last two residues from theC-terminus of purified recombinant human NGF, is 300-fold less efficientin displacing mouse [¹²⁵ I]NGF from the human trkA receptor compared to(1-118)HNGF (49). It is 50- to 100-fold less active in dorsal rootganglion and sympathetic ganglion survival compared to (1-118)hNGF (48).The (1-118)HNGF has considerably lower trkA tyrosine kinaseautophosphorylation activity (49).

NT3 transcription has been detected in a wide array of peripheraltissues (e. g. kidney, liver, skin) as well as in the central nervesystem (e. g. cerebellum, hippocampus) (5); (7), (82). Duringdevelopment, NT3 mRNA transcription is most prominent in regions of thecentral nervous system in which proliferation, migration anddifferentiation of neurons are ongoing (50). Supporting evidence for arole in neuronal development includes the promoting effect of NT3 onneural crest cells (51) and the stimulation of the proliferation ofoligodendrocyte precursor cells in vivo (79). NT3 also supports in vitrothe survival of sensory neurons from the nodose ganglion (NG) (7); (5),(83) and a population of muscle sensory neurons from dorsal rootganglion (DRG) (52). In addition to these in vitro studies, a recentreport showed that NT3 prevents in vivo the degeneration of adultcentral noradrenergic neurons of the locus coerulus in a model thatresembles the pattern of cell loss found in Alzheimer's disease.Currently, there are no published reports concerning the amino acidresidues necessary for trkC binding.

There has been some limited attempts to create chimeric orpan-neurotrophic factors. (See (53); (56); (54), (55)).

SUMMARY OF THE INVENTION

It is an object of the invention to provide pantropic neurotrophins andto produce useful quantities of these pantropic neurotrophins usingrecombinant DNA techniques.

It is a further object of the invention to provide recombinant nucleicacids encoding pantropic neurotrophins, and expression vectors and hostcells containing the nucleic acid encoding the pantropic neurotrophins.

An additional object of the invention is to provide methods forproducing the pantropic neurotrophins, and for treating neuronaldisorders of a patient.

In accordance with the foregoing objects, the present invention providesrecombinant pantropic neurotrophins, and isolated or recombinant nucleicacids which encode the neurotrophins of the present invention. Alsoprovided are expression vectors which comprise DNA encoding a pantropicneurotrophin operably linked to transcriptional and translationalregulatory DNA, and host cells which contain the nucleic acids.

An additional aspect of the present invention provides methods forproducing pantropic neurotrophins which comprises culturing a host celltransformed with an expression vector and causing expression of thenucleic acid encoding the pantropic neurotrophin to produce arecombinant neurotrophin.

Additionally provided are methods of treating a neural disordercomprising administering the pantropic neurotrophins of the presentinvention to a patient.

Additional objects and features of the invention will be apparent tothose skilled in the art from the following detailed description andappended claims when taken in conjunction with the figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the high homology between mouse NGF (SEQ ID NO:1) andhuman NT-3 (SEQ ID NO:2) allows modeling of NT-3 on 3D-structure of NGF.Arrows indicate β-strands. Designations of β-strands as in McDonald etal. (1991) (59). Amino acids which differ between NGF and NT-3 are ingray boxes. Sections with disordered structure are hatched.

FIGS. 2A, 2B, 2C, 2D and 2E depicts the biological effects of selectedmutants on survival of dorsal root ganglion neurons and neuriteextension on PC12/trkC cells. E9 chick DRG neurons cultured for 72 hoursin the presence of conditioned media of 293 cells containing NT-3 ormutant proteins. Response induced by mutants expressed as % of NT-3response. A) 5 ng/ml of NT-3, R103A/D105A and R103A. B) 1 ng/ml NT-3,R103M, R103K, N1, Y51A. C) 0.2 ng/ml NT-3, Y11A, T22Q and K80A Q83A. Theerror is the SD of triplicate determinations.

The response of medium from mock transfected cells was subtracted fromeach data point and was 23%, 23% and 29% for the 200 pg/ml, 1000 pg/mland 5000 pg/ml experiment, respectively. (D) Response of PC12/trkC cellsinduced by conditioned medium containing either NT-3 or R68A mutant.Percentage of cells with neurites induced by different doses ofneurotrophins. The sum of cells with and without neurites was constantfor NT3 and R68A for all doses. (E) Survival of neurons from DRG.Response induced by NT-3, R68A or R114A/K115A expressed as number ofsurviving cells. Results are the mean value of triplicate determinations±SD. The response induced by mock-transfected conditioned medium wassubtracted from data points and was 20±4 surviving cells.

FIGS. 3A, 3B and 3C depict the binding epitopes of NT3 to its receptorstrkC and gp75. The NT3 model is shown with binding determinants frommonomer A and B are shown in light and dark grey, respectively. (A)Epitope for trkC receptor. (B) Side view of trkC epitope. Positions ofD15 and Y51 relative to trkC binding determinants. (C) Epitope for gp75receptor.

FIGS. 4A, 4B, 4C, 4D, 4E, 4F, 4G, 4H, 4I, and 4J depict the screening ofNT3 mutants for improved BDNF and NGF like activities. PC12 cells or thePC12 cell line expressing trkB were plated on collagen-coated dishes.The PC12/trkB cell line was treated with PC12 medium supplemented eitherwith BDNF (A), NT3 (B), mutant D15A (C) or supernatant of mocktransfected 293 cells (E). The PC12 cells were treated with PC12 mediumsupplemented either with NGF (F), NT3 (G), S1 (H), D15A (D), MNTS-1 (I)or supernatant of mock transfected 293 cells (J). Representative fieldswere photographed three days after treatment.

FIGS. 5A, 5B, 5C and 5D depicts that MNTS-1 binds with high affinity tohuman trkA, trkB and trkC. Displacement curves using receptorimmunoadhesins were determined in the presence of a constant amount oflabeled neurotrophin (50 pM for trkA, trkB and trkC; 100 pM for gp75)and increasing amounts of unlabeled competitor; (◯) HNGF, (Δ) HBDNF, (□)NT-3, (▴) MNTS-1, (×) S1, (+) D15A. (A) Displacement of ¹²⁵ I-NGF fromhuman trkA. (B) Displacement of ¹²⁵ I-BDNF from human trkB. (C)Displacement of ¹²⁵ I-NT-3 from human trkC (D) Displacement of ¹²⁵I-NT-3 from human gp75.

FIGS. 6A, 6B and 6C depicts the autophosphorylation of trkA, trkB andtrkC induced by MNTS-1. The PC12 variant cells were exposed toneurotrophic factors and mutants at 25 ng/ml for 5 min. at 37° C. andassayed as described in Experimental Procedures. (A) Response of PC12cells upon addition of no factor, NGF, NT-3, S1, D15A, MNTS-1 andsupernatant of mock transfected 293 cells. (B) Response of PC12/trkBcells upon addition of no factor, NGF, BDNF, NT-3 purified, NT-3expressed, D15A and supernatant of mock transfected 293 cells. (C)Response of PC12/trkC cells upon addition of no factor, NGF, NT-3, D15A,S1, MNTS-1 and supernatant of mock transfected 293 cells. Numbers belowneurotrophic factors indicate type of antiserum used forimmunoprecipitation,443 is a pan-trk antiserum, 656 is a trkC specificantiserum.

FIG. 7 depicts that MNTS-1 is as potent as cocktail of NT-3/BDNF/NGF onsurvival of DRG neurons. Dose dependency of survival of neurons fromDRG. Number of cells supported by cocktail of NT3/BDNF/NGF (1:1:1) ()and MNTS (▴). Results are expressed as mean of triplicate determinations±SD. Data was fit to a four-parameter equation for MNTS-1 (dashed line)and cocktail (solid line). The calculated EC-50 values for MNTS-1 andcocktail were 36 pg/ml and 44 pg/ml, respectively.

FIG. 8 depicts the homology, variable regions and constant regions ofthe various neurotrophins. NGF (SEQ ID NO:3), BDNF (SEQ ID NO:4), NT3(SEQ ID NO:2) and NT4/5 (SEQ ID NO:5) are shown, with the variableregions boxed.

FIGS. 9A, 9B and 9C depict the competition displacement of [¹²⁵ I]hNGFfrom either trkA, p75, or trkA+p75 receptors by increasing concentrationof purified N-terminally-modified forms of hNGF. Upper (9A), middle(9B), and lower (9C) panels represent displacement from MH3T3, A875melanoma, and PC12 pheochromocytoma cells expressing trkA, p75, andtrkA+p75 receptors, respectively. The competing ligands are asindicated: (--) 111/111=homodimers of (10-118)hNGF;(◯--◯)118--118=homodimers of (1-118)hNGF; (▴--▴) 115--115=homodimers of(6-118)hNGF;(▾--▾) mouse NGF=homodimers of (1-118)mNGF. Receptor bindingwas performed at 4° C. and analyzed as described in the examples. Thedata presented represents the total binding (specific and non-specific)and is representative of at least three separate binding experiments foreach cell line. The IC₅₀ for this experiment is given in Table 5.

FIGS. 10A and 10B depicts the autophosphorylation of trkA induced byN-terminally truncated forms of hNGF. Upper panel (10A) shows theintensity of the autophosphorylation of the p140^(trkA) band as afunction of molar concentration of hNGF variant while the lower panel(10B) represents quantitation of the optical density of each banddetermined by reflective densitometry. The autophosphorylatedp140^(trkA) band was identified by anti-trkA immunoblotting onnitrocellulose following immunoprecipitation with antiphosphotyrosine.The data presented are representative of two independent experiments.

FIGS. 11A and 11B depicts the expression and protein analysis of hNGFmutants. Panel A represents an autoradiograph of metabolic labelledmutants 1-8 (See Table 1 for description) separated by SDS-PAGE. Mutantswere transiently expressed in human 293 cells and labelled with ³⁵S-methionine and cysteine. Conditioned media was immunoprecipitated witha purified rabbit anti-hNGF polyclonal antibody, and the precipitatesanalyzed by SDS-PAGE as described in the examples. The lanes labelled wtor B represents transfection of cells with wild type (1-120)hNGFexpressing vector or AdVA vector alone, respectively. Panel B representsWestern immunoblot analysis of approximately 0.1 μg of non-labelledmutant or wild type hNGF. These samples are taken from conditioned mediafollowing transfection in parallel to the metabolic-labelled cellsdescribed above. Immunoblotting was performed with the same anti-hNGFpolyclonal antibody described in panel A. The results can be contrastedwith the variable detection of the mutants immunoblotted in parallel inthe same experiment and reacted with a specific hNGF monoclonal antibodyas shown in FIG. 18. The lane labelled NGF represents the signal from0.1 μg of purified (1-120)hNGF. The left axis of both panels indicatesthe relative mobility of the molecular mass markers in kD.

FIGS. 12A, 12B, 12C, 12D, 12E and 12F depict the competitiondisplacement of [¹²⁵ I]hNGF from cells expressing trkA (top panels, 12Aand B), p75 (middle panels, 12C and 12D), and p75+trkA (bottom panels,12E and 12F) by increasing concentrations of hNGF mutants. For claritythe data is divided into two panels for each cell line. For comparisonsof relative binding affinity, four mutants and the hNGF wild typecontrol were tested in each cell line in one experiment. Each of the twopanels is representative of at least two separate binding experimentsfrom one transfection and one binding experiment from an additionaltransfection. The binding experiments were performed at 4° C. asdescribed in the examples. Total binding is presented as in FIG. 12. TheIC₅₀ relative to wild type hNGF is presented in Table 5.

FIGS. 13A and 13B depict the autophosphorylation of trkA elicited byhNGF mutants. The top panel (13A) represents an autoradiograph ofp140^(trkA) following stimulation of trkA-expressing cells by theindicated concentrations of mutant or wild type hNGF. Levels of trkAautophosphorylation were determined as in FIG. 10. The bottom panel(13B) is the densitometric quantitation of the above autoradiograph. Thedata presented is representative of at least two experiments comparingthe trkA autophosphorylation elicited by all of the mutants within oneexperiment, and is consistent with data from other experiments comparing3-4 mutants per experiment with hNGF.

FIG. 14 depicts the biological activity of hNGF mutants determined byPC12 cell neurite outgrowth. PC12 cells were grown in the presence ofthe indicated concentrations of mutant or wild type hNGF for 48 hrs. Thepercentage of the total cells within a given microscope field extendingneurites is presented normalized to the maximal response elicited by(1-118)hNGF as described in the examples. The values presented are theaverage of at least two determinations per mutant.

FIGS. 15A, 15B, 15C, 15D, and 15E depict the characterization ofpurified N-terminal region mutants. A. Silver stain of SDS-PAGE (15%acrylamide) of 1 μg of purified H4D mutant 2 (lane 1), purifiedhNT3/hNGF N-terminal chimeric mutant 6 (lane 2), partially purified(1-120)hNGF (lane 3-N), and molecular mass markers in kD (lane 4-M).Details of the purification and analysis presented in the Materials andMethods section. B. Competition displacement of [¹²⁵ I]hNGF by purifiedH4D mutant 2, hNT3/hNGF N-terminal chimeric mutant 6, and purified(1-120)hNGF. Top panel (15B) represents binding to NIH3T3 cellsexpressing trkA, bottom panel (15D) shows binding to A875 cells (p75).C. Top panel (15C) represents concentration dependence (M) ofp140^(trkA) autophosphorylation detected by antiphosphotyrosineimmunoblot as in FIGS. 10 and 13. Bottom panel (15E) presentsdensitometric quantitation of the immunoblot data.

FIGS. 16A and B depict the monoclonal antibody interaction with theN-terminal region of hNGF. A. Immunoblot of 0.1 μg of mutants 1-6 and 8(mutant 7 omitted because of low concentration), hNGF (wt) and controltransfected conditioned media (B). Conditioned media was applied toSDS-PAGE, immunoblotted onto nitrocellulose, and reacted with anti-hNGFmonoclonal antibody 14.14. The relative mobility of the mutants is shownas 14 kD. B. Competition displacement of 25 pM[¹²⁵ I]hNGF from eithertrkA-expressing or p75-expressing cells by increasing concentrations ofthe same monoclonal antibody used in panel A.

FIG. 17 depicts a schematic drawing of a hNGF monomer, based on thex-ray crystal structure of murine NOF, which indicates the primary aminoacid sequence (SEQ ID NO:3), the basic features of the secondarystructure and residues modified by mutagenesis. The yellow shadedresidues indicate those which differ between hNGF and hNT3, and werereplaced by the corresponding hNT3 residues in hNGF as domain swaps. Thelarge black numbers located near blocks of 5-8 yellow residues numeratethe particular neurotrophin variable region. These variable regions alsoinclude the amino and carboxy termini. The red shaded residues indicatethose mutated singly or in pairs, and represent amino acids mostlyexposed to the solvent.

FIGS. 18A, 18B and 18C show the hNGF organization. FIG. 18A depicts theposition of variable domains within the primary sequence of hNGF. FIG.18B depicts the variable domain chimeric mutants of hNGF, containing asingle variable domain of hNT3. The list, 18C, contains the specificresidues of hNGF replaced by hNT3 within a given mutant

FIGS. 19A and 19B depict the characterization of a new receptor bindingprocedure used to analyze structural variants of hNGF. A) A comparisonof the binding properties of trkA-IgG immunoadhesion-based assays withthose of holo-trkA receptors expressed in NIH3T3 cell lines. ThetrkA-IgG competition binding profiles are very similar to those ofholo-trkA cell lines (19A) while trkA-IgG displays the same neurotrophinselectivity (19B; NGF>>NT3>BDNF). Bnding data now presented utilizesindividual trk A, B, C or p75-IgG immunoadhesion assays. The receptorbinding characteristics for several variants was verified in holo-trkAcell binding assays.

FIGS. 20A and 20B depict the binding of hNGF/hNT3 chimeric mutants totrkA-IgG and gp75 receptors. The relative affinities of the mutants areplotted as the ratio of the IC₅₀ of the mutant to that of hNGF takenfrom competition binding curves. The average ratio is presented fromthree independent binding experiments. The NGF/NT3 N-terminal domainswap mutant (mutant 6) results in a significant loss of trkA bindingwhile gp75 binding is unaffected. These results are consistent with thedata obtained from holo-trkA binding in cells at 4° C. (FIG. 18B,C).Chimeric mutants containing the first beta-turn of NT3 (mutants 10 and19) have lost 4-fold potency in binding to gp75. Alanine replacement ofbasic residues in the first beta-turn (mutant 21) or in the C-terminus(mutant 24) results in significant loss of gp75 binding.

FIGS. 21A and 21B depict the ability of the hNGF/hNT3 chimeric mutantsto elicit trkA tyrosine kinase autophosphorylation and PC12 cell neuriteoutgrowth, respectively. TrkA-expressing CHO cells were stimulated withhNGF, hNT3 or hNGF/hNT3 domain-swapped chimeric mutants andautophosphorylation was determined by a phosphotyrosine-ELISA assay(OD_(450/650)). Consistent with the trkA binding, little trkAautophosphorylation is stimulated by the N-terminal hNGF/hNT3 chimericmutant. A 2-3- fold loss of activity results from the domain swap withinthe pre-beta turn 1 region (V18, V20, G23), indicating a possible roleof these residues in determining NGF-trkA specificity. For PC12 celldifferentiation, the EC₅₀ for neurite outgrowth was determined for allmutants and expressed as a ratio with the EC₅₀ for hNGF. Again, thegreatest effect is observed with the N-terminal hNGF/hNT3 domain swapmutant, however, loss of bioactivity is also observed with the pre-betaturn 1 region, consistent with the trkA binding and autophosphorylation.

FIGS. 22A and 22B depict the pan-neurotrophic activity of hNGF/hNT3domain-swap mutants. The activity is measured by trkC-IgG competitionbinding and neurite outgrowth in trkC-transfected PC12 cells.Competition displacement of [¹²⁵ I]hNT3 is observed only for hNT3 (IC₅₀=45 pM) and the variable region 4 hNGF/hNT3 domain swap mutant (Mutant16; IC₅₀ =80 nM). Similarly, the hNGF/hNT3 domain swap in variableregion 4 results in significant neurite outgrowth in trkC-transfectedPC12 cells that do not respond to hNGF. Comparison of the trkA-dependentbinding, autophosphorylation, and PC12 cell activities for mutant 16(FIGS. 19, 20) reveals little loss of endogenous hNGF-like activity. TheN-terminal domain-swap mutant (mutant 6), containing the N-terminus ofNT3, does not interact with trkC.

FIG. 23 depicts the two N-terminal domain-swap mutants: hNT3-NH2/hNGF ismutant 6 and contains hNT3 residues 1-6 (YAEHKS-), replacing hNGFresidues 1-7, on a remaining backbone of hNGF. hNGF-NH2/NT3 is P2 (S1)and contains hNGF residues 1-7 (SSSHPIF-), replacing hNT3 residues 1-6,on a remaining backbone of hNT3.

FIGS. 24A, 24B and 24C depict the pan-neurotrophin receptor bindingactivity of P2 (S1) with trkA, trkC, and gp75, respectively. Competitionbinding assays were performed by displacement of [¹²⁵ I]hNGF or [¹²⁵I]hNT3 from the receptor-IgG immunoadhesions by the indicated factor. P2(NGF-NH₂ /NT3), the N-terminal domain-swap mutant of hNT3 containing thehNGF N-terminus, displays hNGF-like trkA binding activity whileretaining trkC activity. NT3-NH₂ /NGF (mutant 6), the converseN-terminal domain-swap mutant of hNGF containing the hNT3 N-terminus,displays reduced trkA binding and no binding to trkC.

FIGS. 25A and 25B depict the pan-neurotrophin bioactivity of P2 (S1) innormal trkA expressing PC12 cells and trkC-transfected PC12 cells withreduced hNGF response, respectively. The percent of PC12 cells bearingneurites at a given concentration of neurotrophin or variant wasnormalized to the maximal response elicited by the natural neurotrophin.The EC₅₀ for P2 (NGF-NH₂ /NT3) in the trkA PC12 cells is 0.4 ng/ml (15pM) and 0.2 ng/ml (7 pM) in the trkC-transfected PC12 cells, similar tothe EC₅₀ for hNGF and hNT3, respectively.

FIGS. 26A and 26B depict the binding of hNGF point mutants to trkA andgp75 receptors. The affinities of the mutants were determined bycompetition binding and are presented as the ratio of the mutant IC₅₀ tothat of natural hNGF. The IC₅₀ was determined by competition bindingcurves performed at least twice from two independent transfections ofeach mutant hNGF. The SD of the IC₅₀ ratios for 4-6 independentmeasurements for each mutant is presented. The residues of hNGF testedfor effects on binding were mostly surface-exposed, as predicted by themurine NGF crystal structure.

FIG. 27 depicts the biochemical activity of hNGF mutants measured by atrkA-autophosphorylation ELISA assay. The EC₅₀ for each mutant, relativeto that of hNGF (EC₅₀ =120 pM), is presented. Mutated residues havingthe greatest effects on potency are indicated by residue number. Mutatedresidues having additional effects on extent of autophosphorylation(efficacy) are indicated by residue number and asterisk (*).

FIG. 28 depicts the bioactivity of selected hNGF mutants in thetrkA-PC12 cell neurite outgrowth assay. The ratio of the EC₅₀ forneurite outgrowth of each mutant compared to the response of hNGF isplotted. Mutated residues with the greatest effects are indicated. Allother mutants are presently being examined.

DETAILED DESCRIPTION OF THE INVENTION

Single letter codes for the amino acids are used herein, as is known inthe art, according to the following table:

                  TABLE I                                                         ______________________________________                                                       three letter                                                                            single letter                                          Amino acid             abbreviation  abbreviation                           ______________________________________                                        Alanine        Ala       A                                                      Arginine               Arg              R                                     Asparagine             Asn              N                                     Aspartic Acid          Asp              D                                     Cysteine               Cys              C                                     Glutamine              Gln              Q                                     Glutamic Acid          Glu              E                                     Glycine                Gly              G                                     Histidine              His              H                                     Isoleucine             Ile              I                                     Leucine                Leu              L                                     Lysine                 Lys              K                                     Methionine             Met              M                                     Phenylalanine          Phe              F                                     Serine                 Ser              S                                     Threonine              Thr              T                                     Tryptophan             Trp              W                                     Tyrosine               Tyr              Y                                     Valine                 Val              V                                     Proline                Pro              P                                   ______________________________________                                    

Thus, the identification of an amino acid residue is the single letteramino acid code followed by the position number of the residue. It is tobe understood that the position number corresponds to the particularneurotrophin backbone; thus, D15A NT3 means that the aspartic acid atposition 15 of NT3 is changed to an alanine. This aspartic acid, foundwithin a "constant region" as defined below, corresponds to position 16of NGF, since NGF has an additional amino acid at its N-terminus, asshown in FIG. 8.

The present invention provides pantropic neurotrophins. Generally, aneurotrophin is a protein involved in the development, regulation andmaintenance of the nervous system, and in particular of neurons.Currently, there are at least five known important neurotrophic factors:nerve growth factor(NGF), neurotrophin-3 (NT3), neurotrophin-4 (NT4,also sometimes called neurotrophin-5 (NT5) or NT4/5), brain-derivedneurotrophic factor (BDNF), and ciliary neurotrophic factor (CNTF).

By the term "pantropic neurotrophins" or "pantropic neurotrophicfactors", or grammatical equivalents, herein is meant a neurotrophinwhich, unlike naturally occurring neurotrophins, has multipleneurotrophin specificities. That is, it contains domains which conferdifferent neurotrophin specificities. In one embodiment, this means thatthe pantropic neurotrophins of the present invention will bind to avariety of neurotrophic receptors. Thus, for example, naturallyoccurring NGF, which is the natural or native ligand for the trkAreceptor, does not bind appreciably to either the trkB or trkC receptorwith high affinity; for example, NGF binds to these receptors with a500-1000 fold lower KD than BDNF or NT3, respectively. However, apantropic NGF, i.e. a pantropic neurotrophin whose amino acid backboneis based on NGF, may bind to at least the trkA, trkB and p75 receptor.Alternatively, a pantropic NGF will bind to the trkA, trkC and p75receptor. A preferred embodiment allows the binding of the trkA, trkB,trkC and p75 receptor. Similarly, naturally occurring BDNF and NT4/5,which are the natural ligands for the trkB receptor, do not bindappreciably to either the trkA or trkC receptor as above. Thus pantropicBDNF or NT4/S will bind to trkB and any combination of trkA, trkC andp75, as shown above for pantropic NGF.

In alternative embodiments, the naturally occurring neurotrophin willbind with poor affinity to several neurotrophin receptors. In thisembodiment, the pantropic neurotrophin binds to these receptors withaffinities higher than normally found, similar to the affinities seenfor the natural ligand. For example, NT3 binds strongly to trkC, andweakly to trkA and trkB. Thus, a pantropic NT3 binds to trkC with itsnormal binding affinity, and will bind to either trkA with an affinitysimilar to the trkA natural ligand, NGF, or to trkB with an affinitysimilar to the trkB natural ligands BDNF or NT4/5, or both.

In the preferred embodiment, the binding affinity of the pantropicneurotrophin for neurotrophin receptors is at least about 50-60%,preferably about 75-80%, and most preferably about 90% of the bindingaffinity of the natural ligand. Thus, a pantropic NGF will bind to thetrkB or trkC receptor with at least 50% of the binding of BDNF or NT4/5,or NT3, respectively. This affinity is measured by a variety of ways, aswill appreciated by those skilled in the art. The preferred method isthe use of competition assays, as shown in (84) and in Example 2.Generally, binding affinities are reported as IC₅₀, that is, theconcentration of unlabeled competitor which inhibits 50% of the bindingof labeled ligand to the receptor.

In alternative embodiments, the pantropicity of the neurotrophin ismeasured not by binding affinity to neurotrophin receptors, but ratherby the neuronal survival or neurite outgrowth assays. Thus, allneurotrophins support the survival of embryonic neural crest-derivedsensory neurons (77), (78), (7), (17). Survival of embryonic sympatheticneurons is only supported by NGF, while survival of placode-derivedsensory neurons is supported by NT3 and BDNF (85). Survival of sensoryneurons of the dorsal root ganglion is supported by both NGF and BDNF(13). NT3 elicits neurite outgrowth of sensory neurons from dorsal rootganglion, sympathetic chain ganglia, and nodose ganglion, as well assupports survival of nodose ganglia neurons and dorsal root ganglionneurons. Thus, neuronal survival assays or neurite outgrowth assays canbe run to determine the pantropicity of the pantropic neurotrophins.

Thus, neurotrophin specificity is determined by the neurotrophinreceptor binding, and the neuronal survival assays and/or neuriteoutgrowth assays. Thus, a pantropic neurotrophin with NGF specificitymeans a neurotrophin which exhibits at least the bindingcharacteristics, neuronal survival assay specificity, or the neuriteoutgrowth assay specificity of NGF. Similarly, a pantropic neurotrophinwith BDNF, NT3 or NT4/5 specificity exhibits at least the bindingcharacteristics, neuron survival assay specificity, or neurite outgrowthassay specificity of BDNF, NT3 or NT4/5, respectively.

In an additional embodiment, pantropic neurotrophins are made byconstructing covalent heterodimers. Normally, neurotrophins arehomodimers, comprising two identical monomers which are noncovalentlyassociated. In this embodiment, as outlined below, pantropicity isconferred by each monomer containing domains which confer differentneurotrophic specificity. Alternatively, pantropicity may be created bycovalently attaching two different neurotrophins with differentspecificities to create a covalent heterodimer. Thus, for example, a NGFmonomer may be covalently attached to a NT3 monomer, resulting in apantropic neurotrophin with both NGF and NT3 specificity. Similarly,covalent heterodimers may be made with any combination of NGF, NT3,NT4/5, BDNF or CNTF to create pantropic neurotrophins with at least twospecificities. In addition, this procedure may be done with monomerswhich are themselves pantropic, resulting in covalent dimers of anycombination of pantropic and single specificity monomers. Thus, apantropic covalent dimer may be a homodimer of two pantropic monomers.However, to be included within the definition of the present invention,the pantropic covalent dimer must have at least two, and preferablythree, neurotrophin specificities.

The covalent attachment is preferably done as a direct fusion of thenucleic acid, such that when the protein is expressed, the C-terminus ofthe first monomer is attached directly to the N-terminus of the secondmonomer, creating a single nucleic acid encoding the dimer. Inalternative embodiments, a linker may be used, such as short repeats ofclycine, or glycine and serine; for example, a linker such as gly-gly orgly-gly-ser-gly-gly (SEQ ID NO:8) may be used. This is done usingtechniques well known in the art. Other techniques for the covalentattachment of proteins are well known in the art.

Pantropic neurotrophins accomplish pantropic binding, or, as discussedabove, pantropic neuronal survival, by containing domains which conferneurotrophin receptor specificity or binding. A domain may be defined inone of two ways. In the first embodiment, a domain is a portion of theneurotrophin which confers some neurotrophic specificity. In thisembodiment, a single monomer of the pantropic neurotrophin contains oneor several domains which confer different specificities. The domains canrange in size from a single amino acid to about 10-15 amino acids. Thedomain may be comprised of a combination of amino acids from a differentneurotrophin than the host neurotrophin, i.e. a domain from oneneurotrophin may be substituted into a second neurotrophin, conferringpantropicity to the second neurotrophin. Alternatively, the domain mayresult from amino acid substitutions which are not based on homology toexisting neurotrophins, as outlined below. In the preferred embodiment,the domain comprises a continuous sequence of amino acids; that is, asingle stretch of amino acids is replaced. In other embodiments, thedomain may be comprised of discontinuous amino acids; for example,several regions within the neurotrophin may confer specificity, and thusreplacements at several positions within the neurotrophin are necessaryfor pantropicity.

In some embodiments, there is more than one domain within a neurotrophinwhich can confer neurotrophic specificity, which will depend on theparticular neurotrophin. BDNF, for example, has a number of domainswhich appear to confer BDNF specificity. The present invention showsthat a single amino acid change in NT3, from aspartic acid at position15 to an alanine, confers BDNF specificity to NT3. This domain can alsobe imported into the NGF and NT4/5 sequences at the positions thatcorrespond to position 15 in NT3; i.e. position 16 in NGF or position 18in NT4/5. It should be understood that the corresponding amino acids aredetermined by an examination of the alignment of the sequences, as shownin FIG. 8. In addition to this domain, there are other domains withinBDNF which confer BDNF specificity. For example, the substitution of theBDNF sequence from positions 78 to 88 (QCRTTQSYVR) (SEQ ID NO:9), S orfrom positions 93-99 (SKKRIG) (SEQ ID NO:10) may confer BDNF specificity(55).

Similarly, NT3 has a number of domains which may confer NT3 specificitywhen substituted into a different neurotrophin. A number of residues ofNT3 have been shown to be important in NT3 trkC receptor binding as wellas bioactivity assays. Specifically, mutations at positions R103, D105,K80, Q83, E54, R56, T22, Y51, V97, Y11, E7, R8, E10 and R68 allcontribute to NT3 specificity, since mutations at these positions in NT3cause decreases in NT3 activity. Of these, K80, Q83, T22, and V97 arewithin variable regions as shown in FIG. 8, and the rest are foundwithin constant regions. In addition, residues in the vicinity of theresidues may also give NT3 specificity. In some embodiments, changes inthe constant regions may also give NT3 specificity. Alternatively,mutations at positions R31 and E92caused increases in NT3 binding;specifically, R31A and E92A NT3 showed increased trkC binding. Thesemutations can be directly imported into neurotrophins besides NT3, usingthe procedures described below. The amino acids at any of thesepositions may be changed, as outlined below.

NGF has a number of domains which may confer NGF specificity whensubstituted into a different neurotrophin. The N-terminal amino acids ofNGF confer NGF specificity when substituted for the N-terminal residuesof NT3. Specifically, the 7 N-terminal amino acids (SSSHPIF) (SEQ IDNO:11) of NGF may be substituted for the 6 N-terminal amino acids of NT3(YAEHKS) (SEQ ID NO:12), resulting in a pantropic NT3 with NGFspecificity. The exact number of NGF N-terminal residues is not crucial;as shown in the Examples, and particularly in Example 3, the histidineat amino acid position 4 appears to be quite important for NGFspecificity; thus from about 4 to about 10 N-terminal residues may beexchanged although in some embodiments, a single amino acid change willbe sufficient. Similarly, a number of other residues of NGF have beenshown to be important in NGF trkA receptor binding as well asbioactivity assays. For example, there are a number of residues which,when mutated, lose NGF activity. This shows the importance of theresidue for NGF specificity. These residues include, but are not limitedto, H4, P5, V18, V20, G23, D30, Y52, R59, R69, H75, Y79, T81, and R103.Of these, D30, R59, Y79, and T81 are in "variable regions", i.e. regionswhich vary between the different neurotrophins, as shown in FIG. 8, withthe remainder in constant regions. In some embodiments, the variableregion residues are more likely to cause NGF specificity, since constantregion residues may be important for general structure andcharacteristics, and may not confer specificity. However, as shown abovefor the D15A mutation, mutations in the constant regions can conferspecificity as well. Furthermore, there are a number of amino acidsubstitutions in NGF which increase NGF binding and/or bioactivity.Accordingly, these substitutions may be imported into other neurotrophinbackbones to confer NGF specificity. These residues include, but are notlimited to, E11, F12, D24, E41, N46, S47, K57, D72, N77, H84, D105, andK115.

Once identified, the residues important in neurotrophin specificity canbe replaced by any of the other amino acid residues using techniquesdescribed in the examples and well-known techniques for site-directedmutagenesis. Generally, the amino acids to be substituted are chosen onthe basis of characteristics understood by those skilled in the art. Forexample, when small alterations in the characteristics are desired,substitutions are generally made in accordance with the following table:

                  TABLE 2                                                         ______________________________________                                                             Exemplary                                                  Original Residue             Substitutions                                  ______________________________________                                           Ala               Ser                                                        Arg                           Lys                                             Asn                           Gln, His                                        Asp                           Glu                                             Cys                           Ser                                             Gln                           Asn                                             Glu                           Asp                                             Gly                           Pro                                             His                           Asn, Gln                                        Ile                           Leu, Val                                        Leu                           Ile, Val                                        Lys                           Arg                                             Met                           Leu, Ile                                        Phe                           Met, Leu, Tyr                                   Ser                           Thr                                             Thr                           Ser                                             Trp                           Tyr                                             Tyr                           Trp, Phe                                        Val                           Ile, Leu                                      ______________________________________                                    

Substantial changes in function or immunological identity are made byselecting substitutions that are less conservative than those shown inTable 1. For example, substitutions may be made which more significantlyaffect: the structure of the polypeptide backbone in the area of thealteration, for example the alpha-helical or beta-sheet structure; thecharge or hydrophobicity of the molecule at the target site; or the bulkof the side chain. The substitutions which in general are expected toproduce the greatest changes in the polypeptide's properties are thosein which (a) a hydrophilic residue, e.g., seryl or threonyl, issubstituted for (or by) a hydrophobic residue, e.g., leucyl, isoleucyl,phenylalanyl, valyl or alanyl; (b) a cysteine or proline is substitutedfor (or by) any other residue; (c) a residue having an electropositiveside chain, e.g., lysyl, arginyl, or histidyl, is substituted for (orby) an electronegative residue, e.g., lutamyl or aspartyl; or (d) aresidue having a bulky side chain, e.g., phenylalanine, is substitutedfor (or by) one not having a side chain, e.g., glycine. In a preferredembodiment, the residues are changed to alanine residues.

Other domains within each neurotrophin may be found using the techniquesdisclosed herein. Specifically, the modelling techniques of Example Iallow the identification of putative specificity sites. In addition,homologue-scanning mutagenesis, random mutagenesis, cassettemutagenesis, may all be used to generate putative pantropicneurotrophins which may then be screened for receptor binding using thetechniques described in the Examples and well-known in the art.

In the context of a covalent heterodimer, a domain may also refer to theentire neurotrophin monomer. Thus, a pantropic covalent heterodimer canbe comprised of a domain which confers NT3 specificity, i.e. the NT3monomer, covalently attached to a domain that confers BDNF specificity,i.e. the BDNF monomer. Similarly, an NT3 monomer may be paired with anNGF monomer, or an NGF monomer may be paired with a BDNF monomer. Inaddition, covalent heterodimers may be made with NT4/5 and CNTF monomersas well. In these embodiments, the domain is large, and generallycomprises most or all of the wild-type neurotrophin amino acid sequence.

In the broadest embodiment, a pantropic neurotrophin binds to at leastthree different neurotrophin receptors. In the preferred embodiment, thepantropic neurotrophin binds to at least four different neurotrophinreceptors.

By the term "neurotrophin receptor" or grammatical equivalents herein ismeant a receptor which binds a neurotrophin ligand. In some embodiments,the neurotrophin receptor is a member of the tyrosine kinase family ofreceptors, generally referred to as the "trk" receptors, which areexpressed on the surface of distinct neuronal populations. The trkfamily includes, but is not limited to, trkA (also known as p140^(trk));trkB (also known as p145^(trkB)); and trkC (also known as p₁₄₅ ^(trkC)).In other embodiments, the neurotrophin receptor is p75^(NGFR), alsocalled p75 or low-affinity nerve growth factor receptor (LNGFR). It isto be understood that other as yet undiscovered neurotrophin receptorsmay also bind the pantropic neurotrophins of the present invention, aswill be easily ascertainable by those skilled in the art.

In a preferred embodiment, the pantropic neurotrophin is a pantropicNT3. In this context, a pantropic NT3 is a pantropic neurotrophin whichhas an amino acid sequence homologous to the amino acid sequence of NT3,with domains which confer other neurotrophin specificities. In thepreferred embodiment, the domains are substituted for NT3 residues; thatis, some number of amino acids are deleted from the NT3 sequence, and anidentical or similar number of amino acids are substituted, conferringan additional specificity. For example, the MNTS-1 (multipleneurotrophic specificities-1) pantropic NT3 comprises the first 7 aminoacids of NGF replacing the 6 N-terminal residues of NT3, plus the D15Asubstitution. The MNTS- I pantropic NT3 has NT3, NGF, and BDNFspecificities, and also binds to the p75 receptor. Other pantropic NT3sare made using minimal changes within the N-terminus. For example, sinceH4 and P5 are conserved among NGFs, and 2 hydrophobic residues inpositions 6 and 7 are conserved, the following variants are made: 1)YASHPIF(SEQ ID NO:13)-hNT3; 2) YAHPIF(SEQ ID NO:14)-hNT3; 3) YASHPIS(SEQID NO:15)-hNT3; 4) YAEHPIF(SEQ ID NO:16)-hNT3; 5) YAQHPIF(SEQ IDNO:17)-hNT3. When the D15A substitution is added, the resultingneurotrophins exhibit NGF, NT3 and BDNF specificity. Alternatively,replacing the variable region 2 or 3 or 4, or combinations, of NT3 withthe corresponding region from NGF gives a pantropic neurotrophin withboth NT3 and NGF specificity.

In a preferred embodiment, the pantropic neurotrophin is pantropic NGF.In this context, a pantropic NGF is a pantropic neurotrophin which hasan amino acid sequence homologous to the amino acid sequence of NGF,with domains which confer other neurotrophin specificities. In thepreferred embodiment, the domains are substituted for NGF residues; thatis, some number of amino acids are deleted from the NGF sequence, and anidentical or similar number of amino acids are substituted, conferringan additional specificity. For example, a pantropic NGF is made with aD16A substitution, which confers BDNF specificity, plus substitutions inthe pre-variable region 1 (V18E+V20L+G23T) and in variable region 4(Y79Q+T8 1 K+H84Q+F86Y+K88R). Alternatively, the substitutions in thepre-variable region 1 can be made with only single amino acidsubstitutions in variable region 4; for example, V18E+V20L+G23T and oneof Y79Q, T81K, H84Q, F86Y, or K88R may be made.

In one embodiment, the pantropic neurotrophin is a pantropic NT4/5. Forexample, NGF specificity may be conferred on NT4/5 by replacing theN-terminal 9 amino acids of NT4/5 with the N-terminal 7 amino acids ofNGF.

In one embodiment, binding to the p75 receptor by the pantropicneurotrophin has been substantially diminished or eliminated. Forexample, as shown in FIG. 26, there are a variety of amino acid residueswhich contribute to p75 binding, in which mutations result in diminishedp75 binding. In NT3, mutations at positions R68, Y11, K73. R1 14, K115,Y51, K73, R31 and H33 and in NGF, mutations at positions F12, I31, K32,K34, K50, Y52, R69, K74, K88, L112, S113, R114, and K115 all result indiminished p75 binding. Since F12, I31, K50, Y52, R69, and K74 are allwithin constant regions of the neurotrophins, as shown in FIG. 8, thesechanges are expected to alter p75 binding in the other neurotrophins aswell. The other residues may be altered as well.

In addition to the amino acid changes outlined above, those skilled inthe art understand that some variability of the amino acid sequence istolerated without altering the specificity and characteristics of theneurotrophin. Thus, pantropic neurotrophins can have amino acidsubstitutions, insertions or deletions compared to the wild-typesequences which do not affect pantropicity but are merely variations ofthe sequence. In some embodiments, these mutations will be found withinthe same positions identified as important to specificity; i.e. in somecases, neutral mutations may be made without changing neurotrophinspecificity.

The pantropic neurotrophins of the present invention can be made in avariety of ways, using recombinant technology. By the term "recombinantnucleic acid" herein is meant nucleic acid in a form not normally foundin nature. That is, a recombinant nucleic acid is flanked by anucleotide sequence not naturally flanking the nucleic acid or has asequence not normally found in nature. Recombinant nucleic acids can beoriginally formed in vitro by the manipulation of nucleic acid byrestriction endonucleases, or alternatively using such techniques aspolymerase chain reaction. It is understood that once a recombinantnucleic acid is made and reintroduced into a host cell or organism, itwill replicate non-recombinantly, i.e., using the in vivo cellularmachinery of the host cell rather than in vitro manipulations; however,such nucleic acids, once produced recombinantly, although subsequentlyreplicated non-recombinantly, are still considered recombinant for thepurposes of the invention.

Similarly, a "recombinant protein" is a protein made using recombinanttechniques, i.e., through the expression of a recombinant nucleic acidas depicted above. A recombinant protein is distinguished from naturallyoccurring protein by at least one or more characteristics. For example,the protein may be isolated away from some or all of the proteins andcompounds with which it is normally associated in its wild type host.The definition includes the production pantropic neurotrophins from oneorganism in the same or different organism or host cell. For example,the protein may be made in the same organism from which it is derivedbut at a significantly higher concentration than is normally seen, e.g.,through the use of a inducible or high expression promoter, such thatincreased levels of the protein is made. Alternatively, the protein maybe in a form not normally found in nature, as in the addition of anepitope tag or amino acid substitutions, insertions and deletions.

Using the nucleic acids of the invention which encode pantropicneurotrophins, a variety of expression vectors are made. The expressionvectors may be either self-replicating extrachromosomal vectors orvectors which integrate into a host genome. Generally, expressionvectors include transcriptional and translational regulatory nucleicacid operably linked to the nucleic acid encoding the pantropicneurotrophin. "Operably linked" in this context means that thetranscriptional and translational regulatory DNA is positioned relativeto the coding sequence of the pantropic neurotrophin in such a mannerthat transcription is initiated. Generally, this will mean that thepromoter and transcriptional initiation or start sequences arepositioned 5' to the pantropic neurotrophin coding region. Thetranscriptional and translational regulatory nucleic acid will generallybe appropriate to the host cell used to express the pantropicneurotrophin; for example, transcriptional and translational regulatorynucleic acid sequences from mammalian cells will be used to express thepantropic neurotrophin in mammalian cells. Numerous types of appropriateexpression vectors, and suitable regulatory sequences are known in theart for a variety of host cells.

In general, the transcriptional and translational regulatory sequencesmay include, but are not limited to, promoter sequences, signalsequences, ribosomal binding sites, transcriptional start and stopsequences, translational start and stop sequences, termination and polyA signal sequences, and enhancer or activator sequences. In a preferredembodiment, the regulatory sequences include a promoter andtranscriptional start and stop sequences.

Promoter sequences encode either constitutive or inducible promoters.Hybrid promoters, which combine elements of more than one promoter, arealso known in the art, and are useful in the invention.

In addition, the expression vector may comprise additional elements. Forexample, the expression vector may have two replication systems, thusallowing it to be maintained in two organisms, for example in mammaliancells for expression and in a procaryotic host for cloning andamplification. Furthermore, for integrating expression vectors, theexpression vector contains at least one sequence homologous to the hostcell genome, and preferably two homologous sequences which flank theexpression construct. The integrating vector may be directed to aspecific locus in the host cell by selecting the appropriate homologoussequence for inclusion in the vector. Constructs for integrating vectorsare well known in the art.

In addition, in a preferred embodiment, the expression vector contains aselectable marker gene to allow the selection of transformed host cells.Selection genes are well known in the art and will vary with the hostcell used.

The pantropic neurotrophins of the invention are produced by culturing ahost cell transformed with an expression vector containing nucleic acidencoding a pantropic neurotrophin, under the appropriate conditions toinduce or cause expression of the pantropic neurotrophin. The conditionsappropriate for pantropic neurotrophin expression will vary with thechoice of the expression vector and the host cell, and will be easilyascertained by one skilled in the art. For example, the use ofconstitutive promoters in the expression vector will require optimizingthe growth and proliferation of the host cell, while the use of aninducible or repressible promoter requires the appropriate growthconditions for induction or derepression.

In a preferred embodiment, the pantropic neurotrophin is purified orisolated after expression. The pantropic neurotrophins may be isolatedor purified in a variety of ways known to those skilled in the artdepending on what other components are in the sample. Standardpurification methods include electrophoretic, molecular, immunologicaland chromatographic techniques, including ion exchange, hydrophobic,affinity, and reverse-phase HPLC chromatography, and chromatofocusing.Ultrafiltration and diafiltration techniques, in conjunction withprotein concentration, are also useful. For general guidance in suitablepurification techniques, see (57). The degree of purification necessarywill vary depending on the use of the pantropic neurotrophin. In someinstances no purification will be necessary.

Appropriate host cells include yeast, bacteria, archebacteria, fungisuch as filamentous fungi, and plant and animal cells, includingmammalian cells. Of particular interest are Saccharomyces cerevisiae andother yeasts, E. coli, Bacillus subtilis, Pichia pastoris, SF9 cells,C129 cells, 293 cells, Neurospora, and CHO, COS, HeLa cells,immortalized mammalian myeloid and lymphoid cell lines. A preferred hostcell is a mammalian cell, and the most preferred host cells include CHOcells, COS-7 cells, and human fetal kidney cell line 293.

In a preferred embodiment, the pantropic neurotrophins of the inventionare expressed in mammalian cells. Mammalian expression systems are alsoknown in the art.

Some genes may be expressed more efficiently when introns are present.Several cDNAs, however, have been efficiently expressed from vectorsthat lack splicing signals. Thus, in some embodiments, the nucleic acidencoding the pantropic neurotrophin includes introns.

The methods of introducing exogenous nucleic acid into mammalian hosts,as well as other hosts, is well known in the art, and will vary with thehost cell used, and include dextran-mediated transfection, calciumphosphate precipitation, polybrene mediated transfection, protoplastfusion, electroporation, encapsulation of the polynucleotide(s) inliposomes, and direct microinjection of the DNA into nuclei.

In one embodiment, pantropic neurotrophins are produced in yeast cells.Yeast expression systems are well known in the art and includeexpression vectors for Saccharoniyces cerevisiae, Candida albicans andC. maltosa, Hansenula polymorpha, Kluyveromyces fragilis and K. lactis,Pichia guillerimondii and P. pastoris, Schizosaccharomyces pombe, andYarrowia lipolytica. The methods of introducing exogenous nucleic acidinto yeast hosts, as well as other hosts, is well known in the art, andwill vary with the host cell used.

In a preferred embodiment, pantropic neurotrophins are expressed inbacterial systems. Expression vectors for bacteria are well known in theart, and include vectors for Bacillus subtilis, E. coli, Streptococcuscremoris, and Streptococcus lividans, among others. The bacterialexpression vectors are transformed into bacterial host cells usingtechniques well known in the art, such as calcium chloride treatment,electroporation, and others.

In one embodiment, pantropic neurotrophins are produced in insect cells.Expression vectors for the transformation of insect cells, and inparticular, baculovirus-based expression vectors, are well known in theart. Materials and methods for baculovirus/insect cell expressionsystems are commercially available in kit form; for example the "MaxBac"kit from Invitrogen in San Diego.

Recombinant baculovirus expression vectors have been developed forinfection into several insect cells. For example, recombinantbaculoviruses have been developed for Aedes aegypti, Autographocalifornica, Bombyx mori, Drosophila melangaster, Spodoptera frugiperda,and Trichoplusia ni.

Once expressed, pantropic neurotrophins are used as neurotrophicfactors. These pantropic neurotrophins may be utilized in variousdiagnostic and therapeutic applications.

The pantropic neurotrophins of the present invention are useful indiagnostic methods of detecting neurotrophin receptors. For example, thepantropic neurotrophins of the present invention may be labelled. By a"labelled pantropic neurotrophin" herein is meant a pantropicneurotrophin that has at least one element, isotope or chemical compoundattached to enable the detection of the pantropic neurotrophin or thepantropic neurotrophin bound to a neurotrophin receptor. In general,labels fall into three classes: a) isotopic labels, which may beradioactive or heavy isotopes; b) immune labels, which may be antibodiesor antigens; and c) colored or fluorescent dyes. The labels may beincorporated into the pantropic neurotrophin at any position. Oncelabelled, the pantropic neurotrophins are used to detect neurotrophinreceptors, either in vitro or in vivo. For example, the presence ofneurotrophin receptors can be an indication of the presence of certaincell types, useful in diagnosis. That is, a subpopulation of certaincell types may be shown by the binding of the labelled pantropicneurotrophin to the cells via the receptors.

Additionally, the pantropic neurotrophins of the present invention areuseful as standards in neurotrophin assays. For example, the activity ofa pantropic neurotrophin in any particular assay may be determined usingknown neurotrophin standards, and then the pantropic neurotrophin may beused in the diagnosis and quantification of neurotrophins.

Furthermore, the pantropic neurotrophins of the present invention areuseful as components of culture media for use in culturing nerve cellsin vivo, since many nerve cell cultures require growth factors. As willbe understood by those skilled in the art, the pantropic neurotrophinsof the present invention can replace other neurotrophic factors whichare frequently used as media components. The amount of the pantropicneurotrophins to be added can be easily determined using standardassays.

The pantropic neurotrophins of the present invention are also useful togenerate antibodies, which can be used in the diagnosis, identification,and localization of neurotrophins or neurotrophin antibodies within anorganism or patient. For example, the pantropic neurotrophins can beused to make polyclonal or monoclonal antibodies as is well known bythose skilled in the art. The antibodies can then be labelled and usedto detect the presence, or absence, of the neurotrophins. Thus,diagnosis of neural disorders associated with neurotrophins may bedetected. Alternatively, the antibodies are detected indirectly, byusing a second antibody. For example, primary antibodies may be made inmice or rabbits, and then labelled anti-mouse or anti-rabbit antibodiesare used to detect the primary antibodies. Either of these methods, aswell as similar methods well known in the art, allow the detection ofneurotrophins in a variety of tissues.

In addition, the antibodies Generated to the pantropic neurotrophins ofthe present invention are also useful for the purification ofneurotrophins and pantropic neurotrophins. Since generally the aminoacid substitutions of the pantropic neurotrophins are small, many immuneepitopes are shared by the neurotrophins and pantropic neurotrophins.Thus, antibodies generated to the pantropic neurotrophins will bindnaturally occurring neurotrophins, and thus are useful in purification.For example, purification schemes based on affinity chromatographytechniques can be used, as are well known in the art.

In the preferred embodiment, the pantropic neurotrophins of the presentinvention are administered to a patient to treat neural disorders. By"neural disorders" herein is meant disorders of the central and/orperipheral nervous system that are associated with neuron degenerationor damage. Specific examples of neural disorders include, but are notlimited to, Alzheimer's disease, Parkinson's disease, Huntington'schorea, stroke, ALS, peripheral neuropathies, and other conditionscharacterized by necrosis or loss of neurons, whether central,peripheral, or motomeurons, in addition to treating damaged nerves dueto trauma, burns, kidney disfunction or injury. For example, peripheralneuropathies associated with certain conditions, such as neuropathiesassociated with diabetes, AIDS, or chemotherapy may be treated using thepantropic neurotrophins of the present invention. Additionally, theadministration of NT3 prevents the in vivo degeneration of adult centralnoradrenergic neurons of the locus coerulus in a model that resemblesthe pattern of cell loss found in Alzheimer's disease (86) In addition,the addition of NT3 has been shown to enhance sprouting of corticospinaltract during development, as well as after adult spinal cord lesions(58). In fact, when NT3 was administered with antibodies which inhibitmyelin-associated growth inhibitory proteins, long-distance regenerationwas seen. Thus, the pantropic neurotrophins of the present invention canbe used in place of NT3 in this application.

In this embodiment, a therapeutically effective dose of a pantropicneurotrophin is administered to a patient. By "therapeutically effectivedose" herein is meant a dose that produces the effects for which it isadministered. The exact dose will depend on the disorder to be treated,and will be ascertainable by one skilled in the art using knowntechniques. In general, the pantropic neurotrophins of the presentinvention are administered at about 1 μg/kg to about 100 mg/kg per day.In addition, as is known in the art, adjustments for age as well as thebody weight, general health, sex, diet, time of administration, drug,interaction and the severity of the disease may be necessary, and willbe ascertainable with routine experimentation by those skilled in theart.

A "patient" for the purposes of the present invention includes bothhumans and other animals and organisms. Thus the methods are applicableto both human therapy and veterinary applications.

The administration of the pantropic neurotrophins of the presentinvention can be done in a variety of ways, including, but not limitedto, orally, subcutaneously, intravenously, intracerebrally,intranasally, transdermally, intraperitoneally, intramuscularly,intrapulmonary, vaginally, rectally, or intraocularly. The pantropicneurotrophins may be administered continuously by infusion into thefluid reservoirs of the CNS, although bolus injection is acceptable,using techniques well known in the art, such as pumps or implantation.In some instances, for example, in the treatment of wounds, thepantropic neurotrophins may be directly applied as a solution or spray.

The pharmaceutical compositions of the present invention comprise apantropic neurotrophin in a form suitable for administration to apatient. In the preferred embodiment, the pharmaceutical compositionsare in a water soluble form, and may include such things as carriers,excipients, stabilizers, buffers, salts, antioxidants, hydrophilicpolymers, amino acids, carbohydrates, ionic or nonionic surfactants, andpolyethylene or propylene glycol. The pantropic neurotrophins may be ina time-release form for implantation, or may be entrapped inmicrocapsules using techniques well known in the art.

The following examples serve to more fully describe the manner of usingthe above-described invention, as well as to set forth the best modescontemplated for carrying out various aspects of the invention. It isunderstood that these examples in no way serve to limit the true scopeof this invention, but rather are presented for illustrative purposes.

EXAMPLES Example 1

Molecular modeling of NT-3 and identification of targets for mutationalanalysis

The coordinates for the three-dimensional structure of mouse NGF wereobtained from N. Q. McDonald and T. L. Blundell. The molecular modelingfor human NT-3 was performed on a Silicon Graphics Iris Workstationusing the interactive program InsightII. The representations of NT-3structures were produced using the program MidasPlus. (University ofCalifornia at San Francisco).

When the three-dimensional structure of mouse NGF (mNGF) becameavailable (59) a rational approach to the structural basis ofneurotrophic function using protein engineering techniques becamepossible. The structure of mNGF consists of a tightly associated dimerof two identical amino acid polypeptide chains. The fold of each monomeris formed by extended segments of twisted anti-parallel β-sheets linkedby turns. The molecule has an elongated shape and provides a flathydrophobic surface that forms the interface of the associated monomers(59). A striking feature of the structure is the arrangement of thedisulfide bonds, now known as the cysteine-knot motif (60). This motifis also found in the otherwise unrelated TGF-β (61); (6()) and PDGF-BB(87). Several regions of the mNGF structure, including the amino andcarboxy termini and the loop between residues 43 and 48 were not welldefined, indicating highly flexible structural elements.

The sequence of human NT-3 (hNT-3) is 56% identical and 70% similar tomNGF (FIG. 1). Sequence differences are clustered in the structurallyundefined N-terminus and in the loop region between residues 43 and 48.The relative position of the cysteine residues is conserved, as in allmembers of the neurotrophin family, suggesting the existence of asimilar cysteine-knot motif in hNT-3. The sequence similarity of hNT-3and mouse NGF suggests that both share the same basic three-dimensionalfold and therefore, mNGF was used as scaffold for the hNT-3 model. Inthe second step of model building, side-chains which differed betweenmNGF and hNT-3 were replaced with the hNT-3 amino acids using theInsightII program (Biosym Technology, San Diego, Calif.). If possible,conformations of hNT-3 side-chains were kept similar to those of MNGF,otherwise they were based on rotamer libraries (62), packing andhydrogen bonding considerations. Finally, the insertion of Asn93 and thesubsequent adjustment of the loop 93-95 were cleaned from a search ofcrystal structures in the Protein Data Bank (63). The final modelconsists of 104 amino acids and does not include the six N-terminal(Tyr1-Ser6), the four C-terminal (Ile116-Thr119) and five loop residues(G44-V48). This model allowed the identification of residues that arelikely to be involved in important structural contexts, which led totheir exclusion from mutational analysis. These residues were eitherinvolved in the interface (W20, F52, Y53, W99, W101), in structurallyimportant hydrogen bonds or hydrophobic contacts (S12, I30, Q50, P62,S83, R100, T106, S107), in disulfide bonds (C15, C57, C67, C79, C108,C110) or were buried in the protein interior (V13, S16, S18, V21, D29,I30, V35, V37, I102, I104). However, in some cases, it will be desirableto alter these residues. In addition Glycine and Alanine residues werenot altered except for Gly 44. In contrast to related studies on themNGF/trkA interaction mainly single residues or pairs of amino acidswere substituted rather than exchanging multiple residues (53) (56) (55)or deleting residues (49). Residues were mostly changed to Alanine (64).In some cases it was possible to model larger amino acids asreplacements into the structure in order to potentially create sterichindrance for the receptor ligand interaction.

The first set of mutations probed both conserved and non-conservedresidues, located mainly in β-strands, that are surface exposed andtherefore potentially involved in binding to the trkC and gp75receptors. The current hypothesis proposed for NGF function (55) is thatdivergent residues located in loops connecting β-strands and the terminiare major determinants for receptor binding and specificity. A secondset of hNT-3 mutants evaluated the importance of these residues tointeraction of hNT-3 and its receptors. The total set of mutants coveredessentially the entire surface of the NT-3 molecule.

Example 2

Generation of specific amino acid substitutions of NT3 and pantropicNT3s

Human NT-3 was previously cloned, sequenced and subcloned into apRK-type vector which allows for production of double and singlestranded DNA in E. coli, as well as expression of mature NT-3 in amammalian system under control of the cytomegalo virus promoter (65).Mutagenesis on this vector was performed according to the method ofKunkel (66) (67). After transformation into the E. coli strain XL1-Blue,colonies were screened for the presence of the desired mutation bysequencing single-stranded DNA using the Sequenase version 2.0 kit (U.S. Biochemical Corp.). The entire sequence coding for the mature NT-3was verified for all positive clones. Double-stranded DNA was isolatedfrom XL-I Blue with the QIAGEN DNA purification kit (Qiagen Inc.,Chatsworth, Calif.). This DNA was subsequently used for transfection ofthe fetal human kidney cell line 293 (68). All other recombinant DNAmanipulations were performed as described (69). Well known techniquesare used to generate the primers for all the mutations. The primer forthe D15A mutation was 5'-GGTCACCCACAAGCTTTCACTGGCACATACCGAG-3'(SEQ IDNO:6), and the primer for the S1 mutant (the N-terminal swap of the 6N-terminal amino acids of NT3 for the 7 N-terminal amino acids of NGF)was 5'-GTACTCCCCTCGGTGGAAGATGGGATGGCTCGAGGACCGTTTCCGC CGTG-3'(SEQ IDNO:7).

Expression of wild-type and mutant neurotrophins

Plasmid DNA containing either the hNT-3 or mutant hNT-3 coding sequenceswas introduced into the human fetal kidney cell line 293 by calciumphosphate precipitation (70). The 75% confluent cells were transfectedwith 10 μg of plasmid DNA per 15 mm cell culture dish and incubated for15 h. in serum containing medium. Then the medium was removed andexchanged by serum-free medium (PSO4) supplemented with 10 mg/lrecombinant bovine insulin, 1 mg/l transferrin and trace elements. Thesupernatant was collected after 48 and 96 hours concentratedapproximately 20-fold with centriprep-10 filtration units (Amicon,Beverly, Mass.) and sterile filtered.

Quantification of neurotrophin mutants

The specific hNT-3 ELISA was based on a Protein A purified polyclonalantiserum from guinea pig (Genentech). Each well of a 96-well plate(MaxiSorp; Nunc, Kamstrup, Denmark) was coated overnight at 4° C. with100 μl of 4 μg/ml antiserum in 0.05M sodium carbonate buffer (pH 9.6).After a 1 h blocking step with blocking buffer (PBS+0.5% BSA+0.01%Thimerosal, pH 7.4), the wells were washed six times with ELISA buffer(PBS+0.5% BSA+0.05% Tween-20+0.01 % Thimerosal, pH 7.4). Purifiedrecombinant hNT-3 or samples of hNT-3 mutants of unknown concentrationswere diluted in ELISA buffer to a volume of 100 μl and added to thewells. The plates were incubated for 2h at room temperature withcontinous shaking. After a wash with ELISA buffer, the wells wereincubated with 100 μl biotinylated anti-hNT-3 antibody (Genentech) for2h and again washed with ELISA buffer. 100 μl of a 1:50000 dilution ofstreptavidin/horse radish peroxidase (Zymed, 43-4323) was added to thewells and incubated for 30 min., followed by a wash step with ELISAbuffer. Finally, the color was developed for 15-20 min. using 100 μl ofa PBS solution containing 0.012% H₂ O₂ and 0.04% o-phenylenediamine. Thereaction was stopped by addition of 50 μl of 4.5 N H₂ SO₄. Theabsorption was read at 490 nm and at 405 nm on a Vmax kinetic microplatereader (Molecular Devices, Palo Alto, Calif.). The standard curve wasdetermined using purified recombinant hNT-3 (Genentech) atconcentrations of 50, 25, 12.5, 6.25, 3.13, 1.56 and 0.78 ng/ml. Thesamples with unknown NT-3 concentration were serially diluted 1:10,1:30, 1:90, 1:270, 1:810, 1:2430, 1:7290 and 1:21870 in order to obtainmultiple data-points per sample. The standard curve was determined usinga four-parameter fit of the data points obtained from the assay of thestandard protein.

The amounts of NT-3 mutants after concentration varied between 120 ng/mland 36 μg/ml. The ELISA assay did not detect any NT-3 in supernatantsfrom mock transfected cells nor did it crossreact with recombinant humanNGF from supernatants of NGF transfected cells (data not shown). Foreach set of expressions of NT-3 mutants a native hNT-3 expression wasperformed and quantified by ELISA in parallel in order to obtain acomparative wt concentration for receptor binding studies. All mutantswere expressed, quantified and assayed at least twice.

Iodination

Purified recombinant hNT-3, hBDNF and hNGF (Genentech) were labeled bylactoperoxidase treatment using a modification of the Enzymobeadradioiodination reagent (Bio-Rad) procedure (71). Usually, 2 μg of theneurotrophins were iodinated to specific activities ranging from3000-3500 cpm/fmol. The labeled material was stored at 4° C. and usedwithin 2 weeks of preparation.

Binding assays

Cell based binding assays made use of preparations of membranes fromstable cell lines expressing rat trkC (NIH3T3/trkC, (26)). Competitivedisplacement assays were performed as described previously (26). Mutantswere assayed for binding affinity to the trkC receptor twice for each ofthe multiple expressions with a duplicate set of data points. Thisprocedure allowed estimation of the error of affinity determination foreach of the mutants. Unpurified recombinant NT-3 from transientlyexpressing cells was compared with purified NT-3 for its ability todisplace 125-I labeled NT-3 from trkC receptors expressed on NIH/3T3cells. Both displaced labeled NT-3 with similar IC-50: 7 pM and 9 pM forunpurified NT-3 and pure NT-3. This indicated that unpurified NT-3 fromsupernatants of expressing 293 cells could be quantified precisely andsubsequently used for receptor binding studies. The specificity of thebinding assays was demonstrated by the inability of NGF, BDNF andsupernatant of mock transfected cells to displace bound labeled NT-3from trkC (data not shown).

Receptor immunoadhesin proteins were constructed using human trkA, trkB,trkC and gp75 extracellular domains fused to immunoglobulin constantdomains (Genentech, unpublished results). A 96-well plate (Coming, ELISAwells strips) was coated with 100 pi of 5 μg/ml goat F(ab')₂ anti-humanFc IgG (Organon Technika, West Chester, Pa.) in coating buffer for 15hat 4-8° C. The wells were aspirated, washed 3 times with PBS andincubated for 2 h with 100 μl of a 40 ng/ml solution of the receptorimmunoadhesin protein in binding buffer (Leibovitz's L- 15 mediumsupplemented with 5 mg/ml BSA (Intergen, Purchase, Pa.), 0.1 mg/ml horseheart cylochrome C (Sigma) and 20 mM HEPES, pH7.2). After a wash stepwith PBS, 50 μl of binding buffer was immediately added to the wells inorder to prevent drying. Each of the native and mutant protein stocksolutions was serially diluted, using binding buffer, to give aconcentration range of 4096-2 pM. 25 μl of serial dilution was added perwell, followed by 25 μl of labeled neurotrophins. The finalconcentration of labeled neurotrophins in each well was approximately 50pM for trkA, trkB and trkC assays and 100 pM for gp75 binding assays.After 3 h of incubation at room temperature, the wells were washed withPBS+0.5% Tween-20 and the bound radioactivity was counted. Alldisplacement experiments were analyzed by applying a four-parameter fitprocedure on the data set with the Kaleidagraph software package. Allbinding results in bar graphs are expressed as IC-50 mut/IC-50 wt.

Stimulation of autophosphorylation of trk receptors on PC12 cell linesby neurotrophic factors

Approximately 1×10⁷ cells were treated at 37° C. for 5 min with 25 ng/mlneurotrophin. NP-40 plate lysis and immunoprecipitation with antiserum443 (pan-trk) or 656 (trkC specific) was done as previously described(26). The phosphotyrosine content was analyzed by Western transfer usingmonoclonal antibody 4G10 as previously described (23). 4G10 was detectedas previously described (26).

Differentiation assays on PC12 and PC12 cells expressing trkB and trkC.

Approximately 10³ PC12 cells expressing the different trk family members(trkC; (26) trkB; Soppet, unpublished observations), were plated into 35mm collagen-coated tissue culture dishes containing a total of 2 ml ofmedium. PC12 cells expressing trkC were assayed at three differentconcentrations (10 ng/ml, 1 ng/ml, 100 pg/ml) and the parental PC12cells expressing only trkA or PC12 cells expressing trkB were treatedwith 10 ng/ml of NT-3 mutant supernatants. For each treatment, at least200 cells were counted. The proportion of neurite-bearing cells wasdetermined by counting the number of cells containing processes at leasttwice the length of the cell body after 3-4 days.

Dissection of embryonic tissues and neuronal cultures.

Chick embryos at different stages of development were obtained byincubating white Leghom chick egos (SPAFAS, Reinholds, Pa.) at 38° C. inan egg incubator for the required time. Dorsal root ganglia, nodoseganglia from embryonic day 8 (E8), and sympathetic ganglia fromembryonic day 11 (E11), were dissected in Leibowitz-15 (L-15) mediacontaining 1× penicillin/streptomycin using watch-maker's forceps andelectrolytically sharpened tungsten needles. Embryonic chicken gangliawere trypsinized at 37° C. for 20 min and then washed in culture medium(F14 with 10% heat-inactivated horse serum and 5% heat-inactivated fetalcalf serum) and were gently triturated with a fire-polished pipette togive a single-cell suspension. Chick embryo cells were plated onto 35-mmdishes that had been coated with polyornithine (0.5 mg/ml in 0.15 Mborate buffer at pH 8.6, overnight) and laminin (20 ml/ml for 4-6 hr at37° C.) in 2 ml of culture medium in presence of 2 ng/ml ofneurotrophin, or at the concentrations noted in the text. All cells witha neuronal morphology within a 5×5-mm grid in the center of each dishwere counted 72 hr later.

The results are shown in Tables 3 and 4.

                                      TABLE 3                                     __________________________________________________________________________                           trkC-binding gp75-binding                                                                           Neurite extension                                                                        Phosphorylation          Expression (IC50 mut/IC50 wt) (IC50 mut/IC50 wt) PC12 PC12                 Mutation       (% of wt)                                                                            NIH3T3                                                                              IA      IA       trkC                                                                             trkB                                                                              trkA                                                                             trkC                   __________________________________________________________________________    human NT-3     100    1.00 ± 0.06                                                                      1.00 ± 0.09                                                                        1.00 ± 0.1                                                                          +  -   -  ++                       b-strands                                                                     D15A                        26            0.63 ± 0.20    0.69 ±                                                              0.07    1.00 ±                                                             0.02            +                                                                 +  - ++                                                                    E17A/L19A                                                                              5                                                                        0.95 ±                                                                0.10                                                                              +      -  - ++       T22Q                        42            3.28 ± 0.73    3.55 ±                                                              0.45    0.87 ±                                                             0.04            +                                                                 -  - +                                                                     D23A                                                                                   66                                                                       0.66 ±                                                                0.21                                                                              +      -     -                                                                ++                   K24A                        3             1.44 ± 0.14    1.23 ±                                                              0.15                                                                                     +                                                                  -     -     +                                                                  S25Q                                                                                   114                                                                      0.86 ±                                                                0.13                                                                                  +      -                                                                -     ++                                                                     S26K                                                                                   116                                                                      0.77 ±                                                                0.27                                                                                  +      -                                                                -     ++                                                                     S25K/S26Y                                                                              97                                                                       0.63 ±                                                                0.06                                                                                  +      -                                                                -     ++                                                                     I28Q                                                                                   36                                                                       1.17 ±                                                                0.13                                                                                  +    -                                                                -     N.D.                                                                     T36E                                                                                   269                                                                      0.97 ±                                                                0.26                                                                                  +      -                                                                -     ++                                                                     L38E                                                                                   41                                                                       N.D.                                                                     1.25 ± 0.23                                                                  N.D   N.D   N.D                                                              N.D.                    E40A                        251           0.44 ± 0.08                                                                                 + - - ++                                                                 Y51A                                                                                   4                                                                        >15.00                                                                   21.52 ± 2.32                                                               18.00 ± 1.10                                                                  - - -     +                                                                 Y51F                                                                                   59                                                                       1.25 ±                                                                0.22                                                                                  +      -                                                                -     ++                                                                     E54A                                                                                   3                                                                        2.99 ±                                                                1.62                                                                                1.40 ±                                                               0.05            +                                                                 -     -                                                                   N.D.                     R56A                        11            2.32 ± 0.89                                                                                1.80 ±                                                                0.29            +                                                                 -     -     ++       V63A                        380           1.12 ± 0.13                                                                                0.72 ±                                                                0.01            +                                                                 -     -     ++       R68A                        67            1.46 ± 0.28    1.55 ±                                                              0.56    118.20                                                                ±  ± 46.40                                                                   +/-   -     -                                                                ++                   K80A/Q883A                  12            2.32 ± 0.51    2.72 ±                                                              0.17    1.81 ±                                                             0.06            +                                                                 -     -     +                                                              R87M                                                                                   27                                                                       0.93 ±                                                                0.20                                                                                  +      -                                                                -     ++                                                                     L89E                                                                                   1                                                                        N.D.                                                                     1.18 ± 0.40                                                                  N.D.  N.D.  N.D.                                                             N.D.                    S91M                        49            1.15 ± 0.25                                                                                  +      -                                                                -   + +                 S91E                        85            1.54 ± 0.12                                                                                  +      -                                                                -     ++                S91A/E92A                   35            0.55 ± 0.17                                                                                  +      -                                                               - N.D.                   V97E                        39            1.82 ± 0.35    1.53 ±                                                              0.13    1.34 ±                                                             0.17                                                                           +      -     -                                                               ++                       R103A/D105A                 74            >100.00   130.00 ± 37.0                                                               1.22 ± 0.12                                                                       -     -                                                                - -                      R103A                       426           >100.00   82.00 ± 36.00                                                                 2.19 ± 0.35                                                                       -     -                                                                -  -                   R103M                       100           1.95 ± 0.23    1.89 ±                                                              0.33        1.60                                                              ± 0.10                                                                     +      -     -                                                                +                        R103K                       102           >100.00   117.00 ± 19.00                                                              0.90 ±  ±                                                               0.12             -                                                                -     -     -                                                              D105A                                                                                  75                                                                       0.67 ± 21                                                                              +                                                                 -     -                                                                  ++                       Amino and carboxyl termini                                                    H4A/H7A/R8A/E10A (N1)       461           4.15 ± 0.61     3.70 ±                                                             0.07   1.80 ±                                                              0.30            +                                                                 -     -     ++       NGF-swap (S1)(SEQ I.D. NO:12)  18         N.D.          1.00 ± 0.33                                                              0.82 ± 0.15                                                                       +                                                                     N.D.  +      ++                                                                (YAEHKS >                                                                    SSSHPIF)                 E3A                         251           0.68 ± 0.18                                                                                  +      -                                                                -     ++                H4D                         348           N.D.          1.35 ± 0.13                                                                  N.D   N.D                                                                N.D   N.D.                                                                     E3A/K5A/S6A (N2)                                                                       200                                                                      1.65 ±                                                                0.68                                                                                +      -                                                                -     ++                 Y11A                        101           3.34 ± 0.23     4.17 ±                                                             1.27      57.94                                                               ± 23.91                                                                    +      -     -                                                                ++                       R114A/K115A                 146           1.05 ± 0.12     1.38 ±                                                             0.16    182.66                                                                ±  ± 37.37                                                                     +      -                                                                -     ++                loops and turns                                                               R31A/H33A/Q34A               0             N.D.                                                                                       N.D.  N.D.                                                                 N.D.  N.D.                                                                     R31A                                                                                   74                                                                       0.38 ±                                                                0.12                                                                              8.45 ± 2.18                                                                       +    -                                                                -     N.D.                                                                  H33A                                                                                   81                                                                       N.D.                                                                     1.17 ± 0.02                                                                2.01 ± 0.73                                                                       N.D   N.D                                                              N.D. N.D.                Q34A                        176           1.05 ± 0.05                                                                                   +      -                                                                -     N.D.                                                                   Q34E                                                                                   166                                                                      1.04 ±                                                                0.34                                                                                   +      -                                                                 -     N.D.                                                                 R42A/T43A                                                                               86                                                                      1.28 ±                                                                0.26                                                                                   +      -                                                                 -     ++                                                                   N45A/S46A/K49A/Y51                                                           A           0                                                                       N.D.                                                                      N.D.   N.D.                                                                 N.D.  N.D.                                                                     G44A                                                                                    88                                                                       1.17 ±                                                               0.13                                                                                     +                                                                  -      -     ++                                                                N45A                                                                                    240                                                                      0.88 ±                                                               0.12                                                                                     +                                                                  -      -     ++                                                                S46A                                                                                    265                                                                      0.95 ±                                                               0.22                                                                                     +                                                                  -      -     ++                                                                P47A                                                                                    109                                                                      0.64 ±                                                               0.21                                                                                     +                                                                  -      -     ++                                                                V48A                                                                                    30                                                                       0.62 ±                                                               0.06                                                                                     +                                                                  -      -     ++                                                                K49A                                                                                    31                                                                       1.06 ±                                                               0.21                                                                                     +                                                                  -      -     ++                                                                E59A R61A                                                                               104                                                                      1.21 ±                                                               0.09                                                                                     +                                                                  -      -     ++                                                                K58A/E59A/R61A                                                                          110                                                                      1.68 ±                                                               0.77    1.5 ±                                                              0.39      1.31                                                                ± 0.01                                                                          +      -                                                                 -     ++                 K64A/N65A/D72A              52             1.13 ± 0.26                                                                                     +                                                                  -      -     ++                                                                D71A/H74A/N76A                                                                         5                                                                         0.65 ±                                                               0.20                                                                                0.60 ±                                                               0.06                                                                          +      -      -                                                               ++                       D71A/K73A/H74A              43             0.98 ± 0.37                                                                                5.54 ±                                                               1.85             +                                                                 -      -                                                                 ++                       Q78A                         80            1.24 ± 0.26                                                                                     +                                                                  -      -     ++                                                                N93A/N94A/L96A                                                                          55                                                                       1.01 ±                                                               0.17                                                                                     +                                                                  -      -     ++                                                                K95A                                                                                    122                                                                      1.13 ±                                                               0.34                                                                               1.13 ±                                                                0.13             +                                                                 -      -                                                                 ++                       NGFswap (S2)                 87            0.96 ± 0.09                                                                                     +                                                                  -      -     ++                                                                (ENNKLVG (SEQ ID                                                             NO:19 >                  DGKQAA (SEQ ID NO:20))                                                      __________________________________________________________________________

                  TABLE 4                                                         ______________________________________                                                    DRG        NG                                                        (% of NT-3 + (% of SYMP                                                      Neurotrophin BDNF + NGF)  NT-3 + BDNF) (% of NGF)                           ______________________________________                                        NT-3        12.9 ± 1.9                                                                            41.8 ± 11.6                                                                           1.1 ± 0.5                                  BDNF 35.4 ± 1.6 65.4 ± 19.7 N.D.                                        NGF 53.7 ± 2.8 N.D. 100.0 ± 3.7                                         NT-3 + BDNF 53.7 ± 2.2 100.0 ± 1.8  N.D.                                NT-3 + NGF 66.5 ± 0.6 N.D. N.D.                                            NT-3 + BDNF + NGF 100.0 ± 0.8  N.D. N.D.                                   D15A 11.4 ± 0.4 46.6 ± 10.0 1.4 ± 0.6                                S1 69.3 ± 4.5 46.0 ± 12.4 91.9 ± 5.7                                 MNTS-1 93.0 ± 5.6 86.4 ± 12.4 100.3 ± 6.7                          ______________________________________                                    

Example 3

Generation, purification, and characterization of N-terminal NGFvariants

Several charged and uncharged residues are conserved among NGF proteinsfrom other species. In particular, His4, Pro5, and His8 are conserved in7 of 8 known NGF sequences; Arg9 exists only in human and chicken NGFwhile Met predominates at this position of NGF of other species. Tenmutants were generated by oligonucleotide-directed mutagenesis thateither: 1) replaced some of the charged residues of the N-terminus ofhNGF with alanine individually or together, 2) replaced His4 withnegatively charged aspartic acid which resides in position 3 of theN-terminal hBDNF sequence (65), or 3) generated chimeric hNGF moleculeswhich contained the first 5 or 6 residues of hBDNF or hNT3,respectively, or other variable regions of hNT3. The resulting mutantconstructs were generated in vectors containing a human CMV promoter(70) and were expressed transiently in human 293 cells as describedbelow.

Purified recombinant (1-118), (6-118), and (10-118) were purified fromtransfected CHO cell line conditioned media utilizing reversed-phaseHPLC and high performance ion-exchange chromatography as described byBurton et al. (1992) (48) and Kahle et al. (1992) (49), andcharacterized by N-terminal sequence analysis, SDS-PAGE and amino acidanalysis (data not shown). These processed variants result from in situproteolysis during conditioning of the CHO cell media by as of yetuncharacterized proteolytic enzymes or processing pathways. The purityof each form was 99% based on SDS-PAGE and the concentration wasdetermined by quantitative amino acid analysis. Purification andanalysis of the H4D mutant 2(20 μg from 300 ml media) and the N-terminalhNT3/hNGF mutant 6 (5 pg from 300 ml media) was performed fromserum-free media conditioned by transfected 293 cells (see below) asjust described for the N-terminal truncated variants.

Mutagenesis was performed by the oligonucleotide-directed method (72)with modifications as indicated in the BioRad Muta-Gene kit (66);BioRad, Richmond, Calif.). The mutations were verified by DNA sequencingof single stranded phagemid clones by the chain termination method (73).The hNGF mutants were expressed in conditioned media following transienttransfection of human 293 cells (68) (70). The media used for collectionwas 50:50 F12/DMEM serum-free media containing the N2 supplement and wascollected following 48 hours in the serum-free media. The conditionedmedia was concentrated 10-fold using Amicon concentrators. Theconcentration of hNGF mutants was determined by an enzyme-linkedimmunoassay (ELISA) utilizing purified rabbit anti-hNGF polyclonalantibodies. The concentration of each mutant varied from 3-8 μg/ml. Eachmutant was expressed at least three times and the concentrationdetermined by ELISA 2-3 independent times.

Mutants were also analyzed by metabolic labelling of transfected 293cells (60 mm plates, 1.2 ml media) by the addition of 200 μCi each of ³⁵S-methionine and cysteine (Amersham). After 18 hrs, media is collectedand reacted with either rabbit anti-hNGF polyclonal antibody or mousemonoclonal antibody for 3-4 hrs at 4° C., collected by precipitationwith Protein-A beads (Pharmacia), and applied to 15% acrylamide SDS-PAGEgels (Novex). Following electrophoresis the gels were dried and placednext to X-ray film. Non-radiolabelled mutants were produced as describedabove and 0.1 μg aliquots were lyophilized, redissolved in SDS-PAGEsample buffer, electrophoresed on same gels, and transported ontonitrocellulose according to standard protocols (BioRad). The blot wastreated with rabbit anti-hNGF polyclonal or mouse anti-hNGF monoclonalantibody overnight at 4° C., washed, and mutants detected with alkalinephosphatase-coupled goat anti-rabbit or anti-mouse IgG antibodies.

Receptor Binding, trkA Autophosphorylation, and PC12 Neurite OutgrowthAssays

[¹²⁵ I]hNGF was produced using the Enzymobead method (BioRad), accordingto the method of Escandon (71). The specific radioactivity, determinedby TCA precipitation of aliquots of the starting reaction mixture andgel filtration-chromatographed [¹²⁵ I]hNGF, averaged 60-90 μCi/μg.Receptor binding assays were performed overnight at 4° C. on NIH3T3cells recombinantly expressing rat trkA cells (Kindly supplied by Dr.Luis Parada), p75-expressing A875 human melanoma cells (ATCC), and ratPC12 cells (Kindly supplied by Dr. Louis Reichardt) as described fortrkB-expressin NIH3T3 cells (23). The concentration of NIH3T3-trkA andA875-p75 cells used was 1×10⁶ cells per ml; 5×10⁵ cells per ml for PC12cells. The final concentration of [¹²⁵ I]hNGF was 50 pM in a volume of0.2 ml. The non-specific binding, defined as the [¹²⁵ I]hNGF bound inthe presence of 1×10⁶ M unlabelled hNGF, varied between 15-25% in mostcases for the NIH3T3 trkA cells, 20-35% for the p75-A875 melanoma cells,and 20-30% for the trkA+p75 PC12 cells using the filter binding assay.The data was fitted to a displacement isotherm and an IC₅₀ wascalculated utilizing a 4-parameter equation within the Kaleidagraphprogram. In some instances receptor binding was performed with cells at25° C. for 90 min, and bound [¹²⁵ I]hNGF was separated from free bysucrose cushion-centrifugation.

Autophosphorylation of trkA was performed at 37° C. for 5 min and theextent of phosphorylation was determined by a variation of the methoddescribed by Kaplan (19). Triton X-100 lysed trk A cells wereimmunoprecipitated with agarose bead-immobilized antiphosphotyrosinemonoclonal antibody 4G10 (UBI), electrophoresed on SDS-PAGE (8%acrylamide-Novex), immunoblotted, and probed with rabbit anti-trkApolyclonal antibody (Kindly provided by Dr. David Kaplan). Detection oftrkA was by alkaline phosphatase (AP)-coupled goat anti-rabbit IgGantibody (TAGO). PC12 cells were grown to 20-30% confluency on Primariapolycationic 24-well plates, the media changed to serum-free DMEM highglucose supplemented with N2 containing wild type or mutant variants ofhNGF. After 48 hours, the number of cells projecting neurites longerthan two cell bodies were counted in a representative visual field andexpressed as a percentage of the total cells within the field, usually100-140 cells. The activity of each mutant and NGF control wasdetermined at least twice in separate experiments. The percent ofresponsive cells at maximal concentrations of hNGF varied from 55-75%between experiments with the mean of 63% calculated from 13determinations. To account for the variation in the maximal responsebetween experiments, this mean value was used to normalize all the data.

Inhibition of [¹²⁵ I]hNGF Binding to trkA and p75 Cells by a MonoclonalAntibody to hNGF

Under the same conditions as the filter binding assay described above,increasing concentrations of an anti-hNGF monoclonal antibody were addedto 25 pM [¹²⁵ I]hNGF and incubated for 30 min at 25° C. Then 1×10⁶ cellsper ml of either NIH3T3-trkA or A875-p75 cells were added (0.2 ml finalvolume) and incubated for 4° C. overnight with vigorous mixing. Thesamples were then diluted and filtered on Whatman GF/C filters andcounted.

Results

The results are shown in Tables 5 and 6.

                                      TABLE 5                                     __________________________________________________________________________    Summary of relative receptor binding affinities of hNGF structural            variants                                                                                           Receptor Binding                                         Structural           trk      p75     trk +  p75                              variant Residues changed                                                                           IC.sub.50.sup.a                                                                   mut/NGF.sup.b                                                                      IC.sub.50                                                                        mut/NGF                                                                            IC.sub.50                                                                        mut/NGF                              __________________________________________________________________________    hNGF(1-118)                                                                           None         0.8 1.0  3.0                                                                              1.0  0.5                                                                               1.0                                   hNGF(6-118)    Deletion S1-P5         7.2      9.0      3.0      1.0                                                    1.3       2.6                       hNGF(10-118)   Deletion S1-R9       210.0  265.0     32.0     10.7                                                   41.0       82.0                        mNGF(1-118)    S3T,I6V,R9M,M37T,G40A,  1.8      2.2     4.1      1.4                                                   0.6       1.2                                          K502,D60A,P61S,D65E,                                                          M92T,G94E,V117T,A120G                                       hNGFwt            None                    1.2      1.0      2.0                                                      1.0       0.5       1.0                Mut 1             H4A                     none      >40.sup.c     1.4                                                   0.7   1.8       3.6                 Mut 2             H4D                     >1000.sup.c   ≧1000.sup                                             .c  1.1     0.6      4.3                                                      8.6                                    Mut 3             R9A                     2.0        1.5     1.9                                                     1.0       1.8       3.6                Mut 4             H4A,H8A,R9A               none      >40.sup.c     3.1                                                   1.6    3.9       7.8                                                      Mut 5             S1H,S3D,H4P,P5                                             A,I6(-)  none      >40.sup.c                                                  2.1      1.0    1.7   3.6                                                                        H8R                 Mut 6          S1Y,S2A,S3E,P5K,I6S,F7S >1000.sup.c  >1000.sup.c  3.5                                                 1.8     1.0        2.0                 Mut 7          R59K,D60E,P61A,N62R,        0.5      0.4     1.6     0.8                                                 0.2       0.4                                          D65K,S66N                                                  Mut 8        M92D,D93E,G94N,+N94/95,    1.8      1.3   8.1      4.1                                                   1.4       2.5                                            Q96L,A97V,A98G                                           __________________________________________________________________________

                  TABLE 6                                                         ______________________________________                                        Summary of biochemical and biological activities supported by                    NGF structural variants                                                               trkA autophosphorylation.sup.a                                                    Maximal level at                                                 1 × 10.sup.-8 M PC12 neurite outgrowth.sup.b                          Structural variant                                                                       mutant/hNGF EC.sub.50 ng/ml                                                                          mutant/hNGF                                 ______________________________________                                        hNGF(1-118)                                                                              1.0         0.14       1.0                                           hNGF(6-118)             1.38      0.22      1.6                               hNGF(10-118)            0.38      4.2       30.0                              mNGF(1-118)            1.08     --    --                                      hNGF wt                    1.0  0.20   1.5                                    Mut 1                      0.47      3.1      21.4                            Mut 2                      0.14      5.0      35.7                            Mut 3                    0.92        0.30     2.1                             Mut 4                    0.23        12.0     85.7                            Mut 5                    0.07        16.0     114.3                           Mut 6                    0.07        12.0     85.7                            Mut 7                    0.88        0.24   1.7                               Mut 8                    1.29        0.12     0.9                           ______________________________________                                         .sup.a TrkA autophosphorylation was performed at 1 × 10.sup.-10, 1      × 10.sup.-9, and 1 × 10.sup.-8 M as described in the              Experimentail Procedures and the legends for FIGS. 2 and 5.  The values       represent the ratio of densitometric area of the immmunoblotted               autophosphorylated p140.sup.trkA band following stimulation of NIH3T3trkA     cells by hNGF structural variants versus (1118)hNGF (truncated hNGF) or       wild type hNGF (mutants).                                                     .sup.b PC12 cell differentiation was determined by neurite outgrowth          described in the Experimental Procedures and the legend to FIG. 6. The        EC.sub.50 values and ratios are taken from the data in FIG. 6 and             represent the average from two separate experiments for each mutant.     

To initiate characterization of the N-terminal amino acid residuesnecessary for full hNGF activity, the (6-118) truncated form of hNGF wasisolated from conditioned media of CHO cells recombinantly-expressinghNGF. The nine amino acid truncated form (10-118)hNGF was generated bylimited proteolysis as described (48). The (6-118) and (10-118)hNGF werepurified by high-performance ion-exchange chromatography (HPIEC) andcharacterized by reverse-phase HPLC, N-terminal sequence analysis,SDS-PAGE, and amino acid analysis (Data not shown).

The relative potency of purified (6-118)hNGF to displace [¹²⁵ I]hNGFfrom cell lines expressing trkA, p75, and trkA+p75 were then compared tothose of (10-118)hNGF, (1-118)or(1-120)hNGF, and (1-118)mNGF(FIG. 9). Assuggested by their equivalence in bioactivity (74), initial experimentsindicated no difference in binding properties of (1-118) versus(1-120)hNGF (not shown). The relative IC₅₀ 's for hNGF and trkA (80-100pM), p75 (2-300 pM), and PC12 cells (50 pM) are within a factor of 2-3to the IC₅₀ and Kd values reported by others (49) (75) (76) (20, 21).Consistent with Vroegop et al., we observe a slightly higher affinity ofhNGF to p75 than commonly reported (IC₅₀ =0.3 nM vs 1-2 nM).

Deletion of the first five amino acids results in a 9-fold loss ofbinding to NIH3T3 cells recombinantly expressing rat trkA while littledifference in binding occurs with p75-expressing A875 human melanomacells (no change) or with PC12 cells expressing trkA+p75 (3-fold). Incontrast, a 265- and 82-fold loss of binding to trkA and PC12 cells,respectively, were observed for (10-118)hNGF compared to (1-118)hNGFwhile a 10-fold loss in binding to p75 occurs. The intermediate potencyof displacement by (10-118)hNGF observed with BC12 cells, relative tothe trkA and p75 cells, suggests contributions by both receptors to theprofile of the displacement isotherm (FIG. 9C). Recombinantly expressedrat trkA cells and radioiodinated human NGF were utilized in the presentstudy whereas a prior study of (10-1 18)hNGF by Kahleet al. (1992) usedhuman trkA-expressing cells and radioiodinated mouse NGF. Thus, similardifferences between (1-118)hNGF and (10-118)hNGF binding are observedregardless whether human or rodent trkA or radiolabelled NGF areutilized during the analysis. Furthermore, (1-1 18)hNGF has 2-3 foldgreater affinity to either human or rat trkA than does (1-188)mNGFwhether radioiodinated mouse or human NGF represents the displaceabletracer. Autophosphorylation of trkA (FIG. 10), and PC 12 celldifferentiation activities (Table 6) of (6-118)hNGF were similar tothose elicited by (1-118)hNGF. However, (10-118)hNGF is at least 10-foldless potent as (1-118)hNGF in trkA autophosphorylation and is 80-foldless potent in stimulating PC12 cell neurite outgrowth (FIG. 10; Table6), consistent with previous results (48) (49). Taken together, theseresults suggest that the first five amino acids of the N-terminus areimportant for full hNGF-trkA binding activity but potent receptoractivation and bioactivity are mostly retained. The additional loss ofthe next four residues appears to be more deleterious to trkA bindingand activation, as well as having some effect on the binding to p75.

The mutant forms of HNGF can be detected by metabolic labelling followedby immunoprecipitation, or immunoblot analysis of non-labelledconditioned media, and are represented as fully-processed polypeptidesof 14 kD (FIG. 11). The concentration of each of the mutants wasdetermined by an ELISA utilizing a polyclonal anti-hNGF antibody.Similar levels of expression, together with the predominant presence ofa single processed species recognized by a polyclonal antibody in threedifferent types of immunoreactivity, suggests that the mutants sharestructural stability similar to that of wild-type hNGF.

The replacement of all three charmed amino acids to alanine (Mut4:H4A+H8A+R9A) resulted in the loss of detectable competitivedisplacement of [¹²⁵ I]hNGF from trkA at 4° C. over a concentrationrange of wild-type hNGF that completely displace the tracer (IC₅₀=1×10⁻¹⁰ M; maximum displacement =1×10⁻⁹ M, FIG. 12, top, Table 5). Theloss of receptor binding also correlates with the at least 10-fold lossof potency and 4-5-fold apparent reduction in efficacy of maximal trkAautophosphorylation. Mutant 4 has a 85-fold lower EC₅₀ of PC 12differentiation relative to (1-118)hNGF (FIG. 14), consistent with thePC12 receptor binding profile which appears to reflect displacementlargely from p75 and a non-displaceable component which may reflectlower affinity binding of the mutant to trkA (FIG. 12, bottom).

His4 and Arg9 variants were then analyzed individually. The N-terminalregion of both hNT3 and hBDNF contains a histidine which suggests thepossibility of a conserved functional role. Replacement of His4 of hNGFby either alanine (mutant 1) or aspartic acid (mutant 2) result indramatic loss of trkA binding, autophosphorylation, and PC12 celldifferentiation (FIGS. 12, 13, 14). As suggested by the variation insequence between hNGF and other NGF species at position 9, the mutationR9A did not have large effects on trkA or p75 activities. However, aslightly lower potency was observed for trkA phosphorylation and PC12cell differentiation (FIG. 12, top, middle, FIGS. 13 and 14). At 25° C.,the PSA and H8A variants displayed a 3-fold and 1.5-fold loss of trkAbinding relative to hNGF, respectively, whereas H4D lost approximately40-fold binding potency; no change in binding to p75 was observed. Allof the above mutants displayed less than 2-fold loss of binding to p75whether at 4° C. or 25° C., suggesting that global structural effectsresulting from the mutagenesis are minimal.

To test whether the specific N-terminal sequence of hNGF is required forneurotrophin interaction with trkA, chimeric mutants (mutants 5 and 6)were generated by replacing the N-terminus of hNGF (SSSHPIF) (SEQ IDNO:11) with that of hBDNF (HSDPA) (SEQ ID NO:18) or hNT3 (YAEHKS) (SEQID NO:12). These mutants would therefore retain the dibasic His8, Arg9residues of hNGF. Even at 10-fold higher concentrations of (1-118)hNGFwhich result in complete receptor displacement at 4° C., the resultingchimeric neurotrophins were unable to displace [¹²⁵ I]hNGF from trkA(FIG. 12A) and are less potent than mutants 1 and 2 in eliciting trkAautophosphorylation activity (FIG. 13). Binding interactions of thesemutants with p75 are indistinguishable from those of (1-118)hNGF whilethe PC12 receptor displacement may be mostly a p75 interaction (FIG. 12,bottom). Similar to the triple alanine mutant 4, the N-terminal chimericmutants were the weakest inducers of PC12 cell differentiation whencompared to all structural variants of hNGF; the IC₅₀ shifted nearly100-fold. These results indicate a requirement for the specificN-terminal sequence of hNGF for high affinity binding and agonistactivity involving trkA but not for binding to p75.

To verify that the N-terminal sequence variants are capable ofrestricting hNGF from high affinity trkA interactions while retainingoverall structure, the H4D mutant 2 and the hNT3/hNGF mutant 6, wereexpressed in large amounts and purified. At the highest concentrationpossible, 2000-fold greater (2×10⁻⁷ M) than the IC₅₀ for (1-118)hNGF(IC₅₀ =1×10⁻¹⁰ M), only 30% and 10% displacement of [¹²⁵ I]NGF occurredfrom trkA at 4° C. for mutant 2 and 6, respectively, while bindingprofiles of p75 were similar (FIG. 15). Consistent with the resultsshown in FIG. 13, the purified mutants were significantly less potentthan (1-118)hNGF in the ability to activate trkA autophosphorylation.These results suggest that overall structural stability is maintainedfollowing either of these amino acid replacements and confirms that theloss of high affinity trkA binding and autophosphorylation are due tothese specific modifications.

Chimeric mutants were also generated to initially compare the role oftwo other variable regions of hNGF as possible determinants of trkAreceptor specificity. Six residues within beta-turn variable region 3(Arg59-Ser66) and seven residues within beta turn variable region 5(Met92-Ala98) were exchanged with the corresponding hNT3 residues inmutants 7 and 8, respectively. Mutant 7 was slightly more potent indisplacing [¹²⁵ I]NGF than hNGF from trkA while mutant 8 bound less wellto p75 (3-5 fold). Otherwise these mutants displayed little differencefrom hNGF in their trkA and p75 binding profiles, ability to supporttrkA autophosphorylation or PC12 neurite outgrowth (FIGS. 12, 13, 14).These results suggest that regions 3 and 5 contribute less to the trkAbinding interaction than does the N-terminus. The lower affinity ofmutant 8 to p75 may represent structural changes around the conservedresidue Lys95, shown to interact with p75 (54).

To determine the relative levels of expression of the M_(r) =14,000fully processed form of the structural variants of hNGF, monoclonal andpolyclonal antibodies to hNGF were tested for their ability to recognizemutants by immunoblotting (FIGS. 11 and 12). When equal quantities ofthe N-terminal mutants expressed in conditioned media wereimmunoblotted, several lost the ability to be recognized by themonoclonal antibody whereas all were recognized by the affinity purifiedpolyclonal antibody. The H4D mutant, and the hBDNF or hNT3 N-terminalchimeric mutants displayed no immunoblot signal whereas the H4A+H8A+H9Amutant was less deleterious (FIG. 16A). H4A or R9A mutations did notaffect antibody binding. The monoclonal antibody was then tested for theability to compete with the binding of [¹²⁵ I]NGF to trkA or p75.Increasing concentrations of antibody were inhibitory to the binding of[¹²⁵ I]NGF to either receptor; an IC₅₀ =1×10⁻⁹ M vs 4×10⁻⁸ M indicatesthat it is 40-fold more effective in blocking binding of hNGF to trkAthan to p75. These results suggest that the N-terminus forms at leastpart of the epitope of the hNGF monoclonal antibody, and the binding ofthe antibody to HNGF blocks its interaction with trkA with relativelyhigh affinity. The weaker inhibition of the binding to p75 suggests thateither a lower affinity epitope outside of the N-terminus may contributeto hNGF-p75 binding contacts, or that steric inhibition of the antibodymay partially interfere with p75 binding. Preliminary studies suggestthat a weaker binding epitope for this antibody does indeed exist in thebeta turn 3 region represented by the hNT3/hNGF chimeric mutant 7. Therole of this region in the binding of hNGF to p75 and trkA is presentlybeing investigated. Although it could be argued that the loss of trkAbinding in the presence of the antibody is due to the binding to asecondary epitope or is due to steric inhibition, the data areconsistent with the preferential loss of trkA versus p75 bindingobserved for several of the N-terminal variants presented above.

Example 4

Generation and characterization of hNGF amino acid variants: hNGF andhNT3 pan-neurotrophins

Identification of target residues for mutational analysis

NGF and its neurotrophin family members NT3, BDNF and NT4/5 shareapproximately 56% sequence identity. The receptor binding specificitymay be determined in part by the amino sequence differences among theneurotrophin family members. These residues may bind directly to the trkreceptor, or function as inhibitory constraints on the trk interactionsof other variable, or conserved residues. Domain-swap mutants of hNGFwere generated between hNGF and hNT3 to test the role of the divergentresidues in determining trk receptor specificity. A comparison ofneurotrophin primary sequences reveals that there are 7 regions of 7-10amino acids each which contain most (80%) of the sequence differences(FIGS. 8, 18A and 18B). Between hNGF and hNT3, 52 of 120 amino acidsdiffer. Forty-one of these 52 differences (79%) occur within the 7divergent regions. Among 9 NGF species including man, birds, snakes andfrogs, 24 of the 52 residues are conserved suggesting a contribution tothe trkA binding specificity. An examination of the x-ray crystalstructure of murine NGF reveals that four of the variableregions/domains are structurally characterized as beta-turns, onevariable region is a beta-sheet, and the last two are the amino andcarboxy termini. Each of the divergent regions contains several chargedand polar side chains accessible to the solvent and capable ofinteracting with receptors. Thirteen chimeric, or domain-swap, mutantswere generated by replacing several residues, or all, of the individualseven variable regions of hNGF with the corresponding domain of hNT3.Two additional regions of less divergence were also replaced:pre-variable region I and 4. Thus a total of 90% of the divergent aminoacid residues were evaluated for their role in determining trkA and trkCspecificity. The chimeric mutants were generated byoligonucleotide-directed mutagenesis and mammalian cell expression asdescribed in example 3.

The selection of individual residues of hNGF for mutagenesis wasdetermined primarily by their position within the x-ray crystalstructure of murine NGF. It was assumed that minimal structural chanceresults from the sequence differences between human and mouse NGF sincethe replaced residues are mostly functionally conserved (10/12).Computer- generated modelling of murine NGF, based on the x-ray crystalstructure coordinates and described in example 1, reveals amino acidswhich project side chains into the solvent and could interact with trkAand gp75 receptors. Some of these include variable residues which weresignificantly modified by the domain-swap mutations, however, manyrepresent residues conserved between hNGF and hNT3 within variable andconserved regions. Residues predicted to have minimal side-chainexposure, such as those implicated in forming the dimeric interface(F12, V14, W21, F49, Y52, F54, W76, T85, F86, W99, F101, T106, A107,V109, V111), hydrophobic interior (V36, V38, F53, I71, A89, I102, andI104), and structurally-dependent and buried hydrogen bonding (Q51, S78,T91, R100), were minimally modified. Of the disulfide bond-formingcysteine residues and glycines and alanines, only A97 was modified.Exceptions were made in the following cases: residues I30 and Y52 whichhave surface side chains although they appear at the dimer interface,residues L39, L90, M92 and A97 which also form a hydrophobic surfacepatch, and D16, K25, D30, E55, K57, R59, R69, D72, H75 which exhibitsome side-chain solvent exposure although hydrogen bonded. Most residueswere changed to alanine (64); in some instances other replacements weremade to maintain structure while testing the role of a specificfunctionality in receptor interactions.

Production and receptor binding characterization of hNGF variants

Mutagenesis, expression, and protein characterization of hNGF variantswas performed as described in example 3. Followingoligonucleotide-directed mutagenesis, all mutants were verified bydideoxynucleotide sequencing. The hNGF mutants were expressed in human293 cells (FIG. 11A, B) and following Amicon concentration (10×) andELISA quantification, most hNGF variants were observed to have beenexpressed at levels similar to normal hNGF controls (5-25 μg/ml), withthe exception of mutants which replaced variable regions 2 or 3 (0.6-1μg/ml). Both of these chimeric molecules result in insertion ordeletions of proline residues, plus other significant changes in sidechain functionality, and thus the low recoveries may reflect structuralinstability. Nevertheless, available quantities permitted thedetermination of binding affinities of the hNGF variants to trkA andgp75 receptors. The binding affinity of each hNGF variant was determinedby competition binding utilizing immunoadhesion constructs of the trkand gp75 receptors (88), and radioiodinated neurotrophins as describedin example 2. Each hNGF variant was expressed in 293 cells at leasttwice and binding experiments performed 2-3 times for each transfection.The relative affinity compared to normal hNGF is expressed as the ratioof the mean IC₅₀ for all determinations of a variant to the IC₅₀ ofhNGF.

TrkA autophosphorylation activity and PC12 cell differentiation bioassay

Biochemical activation of trkA kinase by hNGF variants was determined byassessing trkA autophosphorylation as described in example 3. Aquantitative assay was developed (89) which permits dose-dependentdeterminations of the EC₅₀ for trkA autophosphorylation. A trkA receptorvariant, containing a peptide epitope tag derived from a Herpes simplexsurface protein, was stably expressed in CHO cells in 96 well plates(88). The affinity of the epitope-tagged trkA for hNGF is identical tothat of the normal receptor (88). The cells (duplicate wells for eachconcentration) are stimulated with 8 increasing concentrations of hNGFvariant (10 pM-10 nM) for 10 minutes at 37° C. The cells are lysed withTriton X-100 lysis buffer as described in example 3, and transferred toa plate coated with a monoclonal antibody directed to the epitope tag.After binding, the captured trkA is then reacted with a HRP-conjugatedantiphosphotyrosine monoclonal antibody, and the color reactiondeveloped. The absorbance is then read and plotted versus concentration.The EC₅₀ for hNGF is 100-120 pM. Differentiation of PC12 cells wasperformed as described in example 3, however, cells were first grown orprimed in NGF for 7-10 days. Cells bearing neurites were then harvestedand plated in 24 well dishes in normal growth media, and either in thepresence or absence of hNGF variant. The percentage of cells bearingneurites after 72 hours were quantifyied as described in example 2.hNGF/hNT3 pan-neurotrophic variants were evaluated for hNT3-like trkCbioactivity in trkC-transfected PC12 cells which did not respond to hNGF(Kindly provided by Drs. Pantelis Tsolfous and Luis Parada, NCI).

Results

Mutagenic analysis of variable residues by hNGF/hNT3 chimera

Thirteen chimeric mutants were generated by replacing several residues,or all, of each of the 7 variable regions of hNGF, with thecorresponding region of hNT3 (See FIGS. 8, 18A,B). Two less variableregions, one within beta sheet A and the other within a conservedbeta-turn connecting beta sheets B and C, were also replaced.Competition binding experiments were performed with the hNGF variantsdisplacing [¹²⁵ I]hNGF from trkA or gp75 immunoadhesion fusion proteins.These receptors contain the extracellular domain of trkA or gp75, andthe Fc portion of human IgG. These immunoadhesions bind hNGF withaffinities similar to the holo-trkA and p75 receptors, and display asimilar rank order of affinities for the neurotrophins (FIG. 19A,B). TheIC₅₀ for each hNGF variant was averaged and expressed as a ratio of theIC₅₀ determined for normal hNGF (IC₅₀ HNGF=100 pM; FIG. 23A). The mostsignificant effect on the trkA binding, as previously described (FIGS.12, 15), is a nearly 300-fold loss of binding affinity due to theN-terminal domain swap with hNT3. A 2-3 fold loss of binding is observedfor the three residue chance within pre-variable region 1 (beta sheet A:V18E+V20L+G23T). Less than 2-fold loss of trkA binding is observed forother hNGF variants whereas increased binding is observed for thevariable region 3 chimeric mutant (beta-turn 3) and the C-terminus (FIG.20A). Consistent with the loss of trkA binding affinities, dose-responsecurves for trkA autophosphorylation and PC12 cell differentiation(neurite outgrowth) indicate losses of activity by the N-terminal andpre-variable region 1 variants (FIG. 21A,B). The binding to gp75 isreduced by 5 and 7-fold by the exchange of variable region 1 andpre-variable region 4 of hNT3. respectively (FIG. 20B). A 2-3 fold lossof gp75 binding is also observed for the variable region 5 mutant. Theloss of gp75 binding exhibited by the VI exchange is likely due toexchange of K32 to R, K34 to H, and E35 to Q since alanine replacementsof these residues result in loss of gp75 binding (FIG. 8; (54)). Theseresults indicate that binding interactions of hNGF to trkA and gp75involve some of the variable neurotrophin residues.

The loss of trkA binding and receptor activation suggests that thevariable residues within the N-terminus and pre-variable region 1contribute to trk receptor specificity. This possibility was tested bydetermining receptor binding to trkC-IgG immunoadhesion and neuriteoutgrowth in trkC-transfected PC12 cells which do not respond to NGF.Surprisingly, trkC interactions were not conferred by the N-terminus ofhNT3 (Mutant 6, FIG. 22A,B), however, the four amino acid swap invariable region 4 of hNGF (T81K, H84Q, F86Y, K88R) resulted in asignificant trkC interaction (FIG. 22A,B). The lower trkC affinity andpotency of neurite outgrowth of this variant indicates that otherregions likely contribute to efficient trkC interactions. Pre-variableregion 1 mutant is now being evaluated for trkC interactions, as are thecontributions of the individual residues within variable region 4.However, overlapping mutations within variable region 4 suggest thatmultiple residues of V4 may be necessary for the trk specificity. Forexample, the beta-turn 3/4 variant , exchanging three variable residueswhich overlaps at T81K (S73, Y79Q, T81K) , activates neurite outgrowthin trkC-PC12 cells only 5-10% as well as the variable region 4 mutant(FIG. 22B). Nevertheless, trk function is affected by the alanine mutantY79A+T81A, further implicating the variable residues of this region intrk receptor interactions. The retention of trkA activities by thevariable region 4 mutant suggests that the 4 hNT3 residues arecompatible with trkA binding, however, the equivalent hNGF residues maypose an inhibitory constraint on the interaction of hNGF with trkC.

Although the N-terminal domain of the neurotrophins appears not to be ageneral trk specificity domain, this region of hNGF appears to be amajor determinant of trkA interaction. The replacement of the first sixresidues of NT3 with the first seven residues of hNGF results in apantropic variant which binds and activates both trkA and trkC with highaffinity and potency (FIGS. 24, 25 and 26). Furthermore, it retains highaffinity binding with gp75. Thus it may possible to generate aneffective trkA/trkC pantropic neurotrophin starting with hNT3 andincluding variable regions of hNGF such as the N-terminus, V2, V3, V4,and V5. Conversely, hNGF may be modified to contain similar trkA/trkCpantropic properties by exchanging variable residues within hNT3 betasheet 1 (C1) and V4. Although the hNGF/hNT3 chimera replacing variableregion 2 did not result in gain of trkC activity, is did result in smallloss of trkA binding (1.5 fold). The reciprocal domain swap, replacingvariable region 2 of hNT3 with the corresponding hNGF domain, is nowbeing tested for gain of trkA activity and is a candidate trkA/trkCpantrophin.

Mutagenic anaylsis of individual variable and consented hNGF residues:Structural model of hNGF residues which interact with trkA and gp75.

Using the crystal structure of murine NGF as described above, weevaluated by point mutagenesis 45 residues of hNGF, many of which haveside chain functionalities exposed to the solvent and are capable oftrkA or gp75 interactions. Competition binding analysis reveals that H4,P5A, S13, D30, I31, Y52, R59, R69, Y79, T81, and R103 mutations affecttrkA binding 1.8-10 fold, while mutations of residues E41, K57, D72, N77increase binding 1.5-2 fold (FIG. 27). These results indicate that theseresidues are involved in trkA interactions and suggests that variantscould be generated from both the variable residues (H4, P5, I31, R59,Y79, T81) and conserved residues (S13, D30, Y52, R69, R103) that couldeffect the trk specificity. Mutations in residues F12, I31, K32+K34+E35,K50, Y52, R69, K74, H75, K88, L112, S113, R114, and K115 results in3->50-fold losses in gp75 binding (FIG. 30). In particular, nodisplacement was observed in the presence of 10 nM mutants representingchanges in residues F12, K32+K34+E35, Y52, R69, K88, and R114+K115,suggesting that these residues are critical determinants of gp75binding.

Autophosphorylation analysis using epitope-tagged trkA indicateddecreases in the potency of activation by 1.5-6 fold by mutations inresidues H4, P5, D30, Y52, R69, Y79+T81, and R103 (FIG. 28). Significant(20-60%) decreases in the efficacy of trkA autophosphorylation areobserved for all of these mutations except for residue F12. Theseresults are consistent with the potency of PC12 cell differentiation;2-50 fold decreases in the EC₅₀ of neurite outgrowth are observed formutations in residues H4, F12, D30, Y52, R69, Y79+T81, and R103. OtherhNGF variants are presently being evaluated, including mutations of P5.Interestingly, residues in which 5 mutations minimally affect both thetrkA binding and the potency of trkA autophosphorylation can reduce theefficacy of trkA autophosphorylation. The decrease of trkAautophosphorylation may explain the decreased potency of PC12 celldifferentiation elicited by mutations of R69 and Y79+T81. Alternatively,mutations in R69 greatly reduce p75 binding; the role of p75 in hNGFsignal transduction is presently unclear loss of p75 interaction couldcontribute to a reduced biological effect. This possibility is beinginvestigated with other hNGF variants which decrease hNGF binding togp75.

Residues interacting with the trkA and gp75 receptors were modelled bycomputer-generated on the structure of murine NGF. Two major trkAinteracting regions were found by this analysis: 1) The N-terminus (H4,P5), with unknown crystal structure, and 2) A surface formed by Y79,T81, H84 and R103 of beta sheets C and D. Residues V18, V20, G23, Y52,R59 and R69 of beta sheets A and B make some contributions to anextended surface which would wrap around the beta sheet strands. Nearthe area of Y52 and the beta sheet A residues are D30 and I31 of thesecond protomer. These two residues project relatively little surfacearea into the solvent, however, it is possible that they contribute to acontinuous binding surface formed with the beta-sheet residues.

Two major p75 interacting regions were found: 1) Variable region 1 ofone protomer and beta-sheet B and C of the other protomer, 2) Conservedresidues within the C-terminus and beta-turn 3, also from differentprotomers. In contrast to the trkA-interesting residues within a cleftformed by the pairs of beta sheets, the p75 interacting residues appearto be well exposed. As shown by (54) K32 and K34 project from thevariable region of beta-hairpin turn 1. We find the adjacent residuesK50 and Y52 from the other protomer contribute to p75 binding. K88,which contributes significantly to the p75 binding, is in this regionbut is not highly exposed. The other binding surface is composed of K74(beta-turn 3), R114 and K115 (C-terminus ) of one terminus, and F12, R69from the other protomer.

Other potential pantropic molecules are now being constructed andevaluated base on the mutagenesis analysis presented above. A pantrkA/trkC molecule can be generated by the following changes in hNGF:1)pre-variable region 1 (V18E+V20L+G23T) plus variable region 4(Y79Q+T81K+H84Q+F86Y+K88R); 2) pre-variable region 1 plus minimalresidues replacements of variable region 4. A pan trkA/trkC molecule canbe generated by replacing minimal changes within the first sevenresidues of the N-terminus of hNGF and replacing the first 6 residues ofhNT3. Since H4 and P5 are conserved among NGFs and 2 hydrophobicresidues in positions 6 and 7 are conserved, the following variants havebeen made: 1) YASHPIF(SEQ ID NO:13)-hNT3; 2) YAHPIF(SEQ ID NO:14)-hNT3;3) YASHPIS(SEQ ID NO:15)-hNT3; 4) YAEHPIF(SEQ ID NO:16)-hNT3; 5)YAQHPIF(SEQ ID NO:17)-hNT3. A trkA/trkC pantrophin can be generated byreplacing variable regions 2 or 4 or 5, or combinations of theseelements, of hNT3 with the corresponding regions of hNGF. A trkA/trkBpantrophin can be generated by replacing the first 9 amino acid residuesof hNT4/5 with the first 7 residues of hNGF, or in combination withreplacement of residues within variable region 4 or pre-variable region1.

References

(1): Snider, W. D. & Johnson, E. M. (1989) Ann. Neurol.., 26, 489-506

(2): Barde, Y.-A. (1989) Neuron, 2, 1525-1534

(3): Davies et al., J. Neuroscience, 6, 1897 (1986)

(4): Davies A. M., Trends in Genetics 139-143 (1988)

(5): Maisonpierre, P. C., Belluscio, L., Squinto, S., Ip, N. Y., Furth,M. E., Lindsay, R. M. and Yancopoulos, G. D. (1990) Science 247,1446-1451

(6): Rosenthal A, Goeddel, D. V., Ngyuen, T., Lewis, M., Shih, A.,Laramee, G. R., Nikolics, K., and Winslow, W. (1990) Neuron 4, 767-773

(7): Hohn, A., Leibrock, i., Bailey, K., and Barde, Y.-A., Nature, 344,339-341,1990

(8): Kaisho Y, Yoshimura, K. and Nakahama, K. (1990) FEBS Lett. 266,187-191

(9): Ernfors, P., Ibanez, C. F., Ebendal, T., Olson, L., and Persson, H.(1990) Proc. Natl. Acad. Sci. USA 87, 5454-5458

(10): Jones, K. R. and Reichhardt, L. F. (1990) Proc. Natl. Acad. Sci.USA, 87, 8060-8064

(11): Levi-Montalcini, R. and Angeletti, P. U. (1968) Physiol. Rev., 48,534-569

(12): Thoenen H., Bandtlow, C. and Heumann, R. (1987), Rev. Physiol.Biochem. Pharmacol., 109, 145-178

(13): Barde, Y.-A., Edgar, D. and Thoenen, H. (1982) EMBO J., 1, 549-553

(14): Leibrock, J., Lottspeich, F., Hohn, A., Hofer, M., Hengerer, B.,Masiakowski, P., Thoenen, H., and Barde, Y.-A. (1989) Nature, 341,149-152

(15): Holbook, F. et al., (1991) Neuron, 6, 845-858

(16): Berkemeier, L. R., Winslow, J. W., Kaplan, D. R., Nikolics, K.,Goeddel, D. V. and Rosenthal, A (1991) Neuron, 7, 857-866

(17): Ip, N. Y., Ibanez, C. F., Nye, S. H., McClain, J., Jones, P. F.,Gies, D. R., Belluscio, L., LeBeau, M. M., Espinsosa, R., III, Squinto,S. P., Persson, H. and Yancopoulos, G. D. (1992) Proc. Natl. Acad. Sci.,89, 3060-3064

(18): Martin-Zanca, D., Oskam, R., Mitra, G., Copeland, T. and Barbacid,M. (1989), Mol.Cell. Biol., 9, 24-33

(19): Kaplan, D. R., Martin-Zanca, D., and Parada, L. F. (1991) Nature,350, 158-160

(20): Klein, R., Jing, S., Nanduri, V., O'Rourke, E., and Barbacid, M.(1991a) Cell 65, 189-197

(21): Kaplan, D. R., Hempstead, B., Martin-Zanca, D., Chao, M., andParada, L. F. (1991) Science 252, 554-558

(22): Klein, R., Nanduri, V., Jing, S., Lamballe, F., Tapley, P.,Bryant, S., Cordon-Cardo, C., Jones, K. R., Reichardt, L. F., andBarbacid, M. (1991b) Cell 66, 395-403

(23): Soppet, D., Escandon, E., Maragos, J., Middlemas, D. S., Reid, S.W., Blair, J., Burton. L. E., Stanton, B. R., Kaplan, D. R., Hunter, T.,Nikolics, K. and Parada, L. F. (1991) Cell, 65, 895-903

(24): Squinto, S. P., Stitt, T. N., Aldrich, T. H., Davis, S., Bianco,S. M., Radziejewski, C., Glass, D. J., Masiakowski, P., Furth, M. E.,Valenzuela, D. M., DiStefano, P. S. and Yancopoulos, G. D. (1991) Cell,65, 885-893

(25): Lamballe, F., Klein, R. and Barbacid (1991), Cell, 66, 967-979

(26): Tsoulfas, P., Soppet, D., Escandon, E., Tessarollo, L.,Mendoza-Ramirez, J.-L., Rosenthal, A., Nikolics, K. and Parada, L. F.(1993) Neuron, 10, 975-990

(27): Cordon-Cardo, C., Tapley, P., Jing, S., Nanduri, V., O'Rourke, E.,Lamballe, F., Kovary, K., Klein, R., Jones, K. R., Reichhardt, L. F. andBarbacid, M. (1991), Cell, 66, 173-183

(28): Klein, R., Lamballe, F., Bryant, S., and Barbacid, M. (1992)Neuron 8, 947-956

(28a): Klein, R., Parada, L. F., Coulier, F. and Barbacid, M. (1989),EMBO J., 8, 3701-3709

(29): Ip, N. Y, Stitt, T. N., Tapley, P., Klein, R., Glass, D. J.,Fandl, J., Greene, L. A., Barbacid, M. and Yancopoulos, G. D. (1993)Neuron, 10, 137-149

(30): Johnson, D., Lanahan, A., Buck, C. R., Sehgal, A., Morgan, C.,Mercer, E., Bothwell, M. and Chao, M. (1986) Cell, 47, 545-554

(31): Radeke, M. J., Misko, T. P., Hsu, C., Herzenberg, L. A. andShooter (1987) Nature, 325, 593-597

(32): Loetscher, H., Pan, Y.-C. E., Lahm, H.-W., Gentz, R., Brockhaus,M., Tabuchi, H., and Lesslauer, W. (1990) Cell 61, 351-359

(33): Smith, C. A., Davis. T., Anderson, D., Solam, L., Beckmann, M. P.,Jerzy, R., Dower, S. K., Cosman, D., and Goodwin, R. G. (1990) Science248, 1019-1023

(34): Schall, T. J., Lewis, M., Koller, K. J., Lee, A., Rice, G. R.,Wono, G. H. W., Gatanga. T., Granger, G. A., Lentz, R., Raab. H., Kohr,W. J., and Goeddel, D. V. (1990) Cell 61, 361-370

(35): Mallet, S., Fossum, S., and Barclay, A. N. (1990) EMBO J. 9,1063-1068

(36): Camerini, D., Walz, G., Loenen, W. A. M., Borst, J., and Seed, B.(1991) J. Immunol., 147, 3165-3169

(37): Stamenkovic, I., Clarke, E. A., and Seed, B. (1989) EMBO I. 8,1403-1410

(38): Bothwell, M. (1991) Cell, 65, 915-918

(39): Chao, M. V. (1992) Neuron, 9, 583-593

(40): Connolly et al., J. Cell. Biol. 90:176-180 (1981)

(41): Skaper and Varon, Brain Res. 197: 379-389 (1980)

(42): Yu, et al., J. Biol. Chem. 255:10481-10492 (1980)

(43): Haleqoua, et al., Cell 22:571-581 (1980)

(44): Tiercy et al., J. Cell. Biol. 103:2367-2378 (1986)

(45): Hefti, J. Neurosci. 6:2155 (1986)

(46): Korsching, TINS pp. 570-573 (November/December 1986)

(47): Taylor et al. 1991

(48): Burton, L. E., Schmelzer. C. H., Szonyi, E., Yedinak, C., andGorrell, A. (1992) J. Neurochem. 59, 1937-1945

(49): Kahle, P., Burton, L. E., Schmelzer, C. H. and Hertel, C. (1992)J. Biol. Chem., 267, 22707-22710

(50): Maisonpierre, P. C., Belluscio, L., Friedman, B., Alderson, R. F.,Wiegand, S. J., Furth, M. E., Lindsay, R. M. and Yancopoulos, G. D.(1990b), Neuron, 5, 501-509

(51): Kalcheim, C., Carmeli, C. and Rosenthal, A. (1992) Proc. Natl.Acad. Sci. USA, 89, 1661-1665

(52): Hory-Lee, F., Russell, M., Lindsay and Frank, E. (1993) Proc.Natl. Acad. Sci. USA. 90, 2613-2617

(53): Ibanez, C., Ebendal, T., and Persson, H. (1991) EMBO J. 10,2105-2110

(54): Ibanez, C. F., Ebendal, T., Barbany, G., Murray-Rust, J.,Blundell, T. L., and Persson, H. (1992) Cell 69, 329-341

(55): Ibanez, C. F., Ilag, L. L., Murray-Rust, J., and Persson, H.(1993) EMBO J. 12, 2281-2293

(56): Suter, U., Angst, C., Tien, C.-L., Drinkwater, C. C., Lindsay, R.M. and Shooter, E. M. (1992) J. Neurosci., 12, 306-318

(57): Scopes, R., Protein Purification, Springer-Verlag, NY (1982)

(58): Schnell, L., Schneider, R., Kolbeck, R., Barde, Y.-A. and Schwab,M. E. (1994), Nature, 367, 170-173

(59): McDonald, N. Q., Lapatto. R., Murray-Rust, J., Gunning, J.,Wlodawer, A. and Blundell, T. L. (1991) Nature, 354, 411-414

(60): Schlunegger, M. P. and Gratter, M. G. (1992), Nature, 358, 430-434

(61): McDonald, N. Q. and Hendrikson, W. A. (1993), Cell, 73, 421-424

(62): Ponder, J. W. and Richards, F. M. (1987) J. Mol. Biol., 193,775-791

(63): Bernstein, F. C, Koetzle, T. F., Williams, G. J. B., Meyer, Jr.,E. F., Brice, M. D., Rodgers, J. R., Kennard, O., Shimanouchi, T. andTasumi, M. (1977) J. Mol. Biol., 112, 535-542

(64): Cunningham, B. C. and Wells, i. A. (1989) Science, 244, 1081-1085

(65): Rosenthal, A., Goeddel, D. V., Nguyen, T., Martin, E., Burton, L.E., Shih, A., Laramee, G. R., Wurm, F., Mason, A., Nikolics, K., andWinslow, J. W. (1991) Endocrinol. 129, 1289-1294

(66): Kunkel, T. A. (1985) Proc. Natl. Acad. Sci. USA, 82, 488-10

(67): Kunkel et al. 1987

(68): Graham, F. L., Smiley, J., Russell, W. C., and Nairn, R. (1977) J.Gen Virol. 36, 59-77

(69): Sambrook et al. 1989

(70): Gorman, C. M., Gies, D. R. and McCray, G. (1990) DNA Protein Eng.Tech., 2, 3-18

(71): Escandon, E., Burton, L. E., Szonyi, E., and Nikolics, K. (1993)J. Neurosci. Res. 34, 601-613

(72): Zoller, M. J. and Smith, M. (1983) Methods in Enzymol. 100,468-500

(73): Messing, J., Crea, R., Seeburg. P. (1981) Nucleic Acids Res. 9,309

(74): Schmelzer, C. H., Burton, L. E., Chan, W. P., Martin, E., Gorman,C., Canova-Davis, E., Ling, V. T., Sliwkowskl, M. B., McCray, G.,Briggs, I. A., Nguyen, T. H., and Polastri, G. (1992) J. Neurochem. 59,1675-1683

(75): Vroegop, S., Decker, D., Hinzmann, I., Poorman, R., and Buxser, S.(1992) I. Protein Chem. 11, 71-82

(76): Sutter, A., Riopelle, R. I., Hartis-Wattick, R. M., and Shooter,E. M. (1979) I. Biol. Chem. 254, 5972-5982

(77): Thoenen, H. and Barde, Y. A. (1980) Physiol. Rev., 60, 1284-1325

(78): Lindsay, R. M., Thoenen, H. and Barde, Y.-A. (1985) Dev. Biol.,112, 319-328.

(79): Barres et al 1994, manuscript submitted for publication

(80): Davies et al., (1993), J. Neuroscience 13:4215-4223 (1993)

(81): Shelton et al., (December 1984), Proc. Natl. Acad. Sci. USA81:7951-7955

(82): Shelton et al., (April 1986) Proc. Natl. Acad. Sci. USA83:2714-2718

(83) Rosenthal et al., (1990), Neuron, 4:767-773

(84): Hulme, E. C. and Birdsall, M. J. M., Strategy and Tactics inReceptor Binding Studies, p63-212 in Receptor-Ligand Interactions, Ed.E. C. Hulme

(85): Grotz et al., Eur. J. Biochem. 204:745-749 (1992)

(86): Arenas et al., Nature 367:368-371 (1994)

(87): Oefner et al., EMBO J., 11:3921-3926 (1992)

(88): Shelton et al., (1994), manuscript submitted for publication

(89): Sadick et al., (1994), manuscript submitted for publication

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 20                                          - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 120 amino - #acids                                                (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                               - -  Ser Ser Thr His Pro Val Phe His Met Gly - #Glu Phe Ser Val Cys            1               - #5                  - #10                  - #15          - -  Asp Ser Val Ser Val Trp Val Gly Asp Lys - #Thr Thr Ala Thr Asp                            - #20                  - #25                  - #30          - -  Ile Lys Gly Lys Glu Val Thr Val Leu Ala - #Glu Val Asn Ile Asn                            - #35                  - #40                  - #45          - -  Asn Ser Val Phe Arg Gln Tyr Phe Phe Glu - #Thr Lys Cys Arg Ala                            - #50                  - #55                  - #60          - -  Ser Asn Pro Val Glu Ser Gly Cys Arg Gly - #Ile Asp Ser Lys His                            - #65                  - #70                  - #75          - -  Trp Asn Ser Tyr Cys Thr Thr Thr His Thr - #Phe Val Lys Ala Leu                            - #80                  - #85                  - #90          - -  Thr Thr Asp Glu Lys Gln Ala Ala Trp Arg - #Phe Ile Arg Ile Asp                            - #95                 1 - #00                 1 - #05        - -  Thr Ala Cys Val Cys Val Leu Ser Arg Lys - #Ala Thr Arg Arg Gly                           110 - #                115 - #                120             - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 119 amino - #acids                                                (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                               - -  Tyr Ala Glu His Lys Ser His Arg Gly Glu - #Tyr Ser Val Cys Asp             1               - #5                  - #10                  - #15          - -  Ser Glu Ser Leu Trp Val Thr Asp Lys Ser - #Ser Ala Ile Asp Ile                            - #20                  - #25                  - #30          - -  Arg Gly His Gln Val Thr Val Leu Gly Glu - #Ile Lys Thr Gly Asn                            - #35                  - #40                  - #45          - -  Ser Pro Val Lys Gln Tyr Phe Tyr Glu Thr - #Arg Cys Lys Glu Ala                            - #50                  - #55                  - #60          - -  Arg Pro Val Lys Asn Gly Cys Arg Gly Ile - #Asp Asp Lys His Trp                            - #65                  - #70                  - #75          - -  Asn Ser Gln Cys Lys Thr Ser Gln Thr Tyr - #Val Arg Ala Leu Thr                            - #80                  - #85                  - #90          - -  Ser Glu Asn Asn Lys Leu Val Gly Trp Arg - #Trp Ile Arg Ile Asp                            - #95                 1 - #00                 1 - #05        - -  Thr Ser Cys Val Cys Ala Leu Ser Arg Lys - #Ile Gly Arg Thr                               110 - #                115 - #            119                 - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 120 amino - #acids                                                (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                               - -  Ser Ser Ser His Pro Ile Phe His Arg Gly - #Glu Phe Ser Val Cys             1               - #5                  - #10                  - #15          - -  Asp Ser Val Ser Val Trp Val Gly Asp Lys - #Thr Thr Ala Thr Asp                            - #20                  - #25                  - #30          - -  Ile Lys Gly Lys Glu Val Met Val Leu Gly - #Glu Val Asn Ile Asn                            - #35                  - #40                  - #45          - -  Asn Ser Val Phe Lys Gln Tyr Phe Phe Glu - #Thr Lys Cys Arg Asp                            - #50                  - #55                  - #60          - -  Pro Asn Pro Val Asp Ser Gly Cys Arg Gly - #Ile Asp Ser Lys His                            - #65                  - #70                  - #75          - -  Trp Asn Ser Tyr Cys Thr Thr Thr His Thr - #Phe Val Lys Ala Leu                            - #80                  - #85                  - #90          - -  Thr Met Asp Gly Lys Gln Ala Ala Trp Arg - #Phe Ile Arg Ile Asp                            - #95                 1 - #00                 1 - #05        - -  Thr Ala Cys Val Cys Val Leu Ser Arg Lys - #Ala Val Arg Arg Ala                           110 - #                115 - #                120             - -  - - (2) INFORMATION FOR SEQ ID NO:4:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 119 amino - #acids                                                (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                               - -  His Ser Asp Pro Ala Arg Arg Gly Glu Leu - #Ser Val Cys Asp Ser             1               - #5                  - #10                  - #15          - -  Ile Ser Glu Trp Val Thr Ala Ala Asp Lys - #Lys Thr Ala Val Asp                            - #20                  - #25                  - #30          - -  Met Ser Gly Gly Thr Val Thr Val Leu Glu - #Lys Val Pro Val Ser                            - #35                  - #40                  - #45          - -  Lys Gly Gln Leu Lys Gln Tyr Phe Tyr Glu - #Thr Lys Cys Asn Pro                            - #50                  - #55                  - #60          - -  Met Gly Tyr Thr Lys Glu Gly Cys Arg Gly - #Ile Asp Lys Arg His                            - #65                  - #70                  - #75          - -  Trp Asn Ser Gln Cys Arg Thr Thr Gln Ser - #Tyr Val Arg Ala Leu                            - #80                  - #85                  - #90          - -  Thr Met Asp Ser Lys Lys Arg Ile Gly Trp - #Arg Phe Ile Arg Ile                            - #95                 1 - #00                 1 - #05        - -  Asp Thr Ser Cys Val Cys Thr Leu Thr Ile - #Lys Arg Gly Arg                               110 - #                115 - #            119                 - -  - - (2) INFORMATION FOR SEQ ID NO:5:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 130 amino - #acids                                                (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                               - -  Gly Val Ser Glu Thr Ala Pro Ala Ser Arg - #Arg Gly Glu Leu Ala             1               - #5                  - #10                  - #15          - -  Val Cys Asp Ala Val Ser Gly Trp Val Thr - #Asp Arg Arg Thr Ala                            - #20                  - #25                  - #30          - -  Val Asp Leu Arg Gly Arg Glu Val Glu Val - #Leu Gly Glu Val Pro                            - #35                  - #40                  - #45          - -  Ala Ala Gly Gly Ser Pro Leu Arg Gln Tyr - #Phe Phe Glu Thr Arg                            - #50                  - #55                  - #60          - -  Cys Lys Ala Asp Asn Ala Glu Glu Gly Gly - #Pro Gly Ala Gly Gly                            - #65                  - #70                  - #75          - -  Gly Gly Cys Arg Gly Val Asp Arg Arg His - #Trp Val Ser Glu Cys                            - #80                  - #85                  - #90          - -  Lys Ala Lys Gln Ser Tyr Val Arg Ala Leu - #Thr Ala Asp Ala Gln                            - #95                 1 - #00                 1 - #05        - -  Gly Arg Val Gly Trp Arg Trp Ile Arg Ile - #Asp Thr Ala Cys Val                           110 - #                115 - #                120             - -  Cys Thr Leu Leu Ser Arg Thr Gly Arg Ala                                                  125 - #                130                                    - -  - - (2) INFORMATION FOR SEQ ID NO:6:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 base - #pairs                                                  (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                               - -  GGTCACCCAC AAGCTTTCAC TGGCACATAC CGAG      - #                  -      #        34                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:7:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 50 base - #pairs                                                  (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                               - -  GTACTCCCCT CGGTGGAAGA TGGGATGGCT CGAGGACCGT TTCCGCCGTG - #                  50                                                                        - -  - - (2) INFORMATION FOR SEQ ID NO:8:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino - #acids                                                  (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                               - -  Gly Gly Ser Gly Gly                                                        1               - #5                                                        - -  - - (2) INFORMATION FOR SEQ ID NO:9:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino - #acids                                                 (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                               - -  Gln Cys Arg Thr Thr Gln Ser Tyr Val Arg                                    1               - #5                  - #10                                 - -  - - (2) INFORMATION FOR SEQ ID NO:10:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino - #acids                                                  (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                              - -  Ser Lys Lys Arg Ile Gly                                                    1               - #5   6                                                    - -  - - (2) INFORMATION FOR SEQ ID NO:11:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino - #acids                                                  (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                              - -  Ser Ser Ser His Pro Ile Phe                                                1               - #5       7                                                - -  - - (2) INFORMATION FOR SEQ ID NO:12:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino - #acids                                                  (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                              - -  Tyr Ala Glu His Lys Ser                                                    1               - #5   6                                                    - -  - - (2) INFORMATION FOR SEQ ID NO:13:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino - #acids                                                  (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                              - -  Tyr Ala Ser His Pro Ile Phe                                                1               - #5       7                                                - -  - - (2) INFORMATION FOR SEQ ID NO:14:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino - #acids                                                  (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                              - -  Tyr Ala His Pro Ile Phe                                                    1               - #5   6                                                    - -  - - (2) INFORMATION FOR SEQ ID NO:15:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino - #acids                                                  (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                              - -  Tyr Ala Ser His Pro Ile Ser                                                1               - #5       7                                                - -  - - (2) INFORMATION FOR SEQ ID NO:16:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino - #acids                                                  (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                              - -  Tyr Ala Glu His Pro Ile Phe                                                1               - #5       7                                                - -  - - (2) INFORMATION FOR SEQ ID NO:17:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino - #acids                                                  (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                              - -  Tyr Ala Gln His Pro Ile Phe                                                1               - #5       7                                                - -  - - (2) INFORMATION FOR SEQ ID NO:18:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino - #acids                                                  (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                              - -  His Ser Asp Pro Ala                                                        1               - #5                                                        - -  - - (2) INFORMATION FOR SEQ ID NO:19:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino - #acids                                                  (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                              - -  Glu Asn Asn Lys Leu Val Gly                                                1               - #5       7                                                - -  - - (2) INFORMATION FOR SEQ ID NO:20:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino - #acids                                                  (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                              - -  Asp Gly Lys Gln Ala Ala                                                    1               - #5   6                                                  __________________________________________________________________________

We claim:
 1. A polypeptide, comprising NT-3 having an amino acidsubstitution of an aspartic acid corresponding to position 15 in matureNT-3, wherein the substitution confers trkB-binding activity so that thepolypeptide binds to neurotrphin receptors trkB and trkC.
 2. Thepolypeptide according to claim 1, wherein the substitution is alanine.3. A composition comprising the polypeptide of claim 1 and apharmaceutically acceptable carrier.
 4. A covalent homodimer, comprisingtwo D15A NT3 monomers, wherein the homodimer binds neurotrophinreceptors trkB and trkC.